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Sen S, Bhowmik P, Tiwari S, Peleg Y, Bandyopadhyay B. Versatility of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) from diagnosis of early pathological infection to mutation detection in organisms. Mol Biol Rep 2024; 51:211. [PMID: 38270670 DOI: 10.1007/s11033-023-09110-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2023] [Accepted: 12/05/2023] [Indexed: 01/26/2024]
Abstract
Loop-mediated isothermal amplification (LAMP) is a rapid, state-of-the-art DNA amplification technology, used primarily for the quick diagnosis and early identification of microbial infection, caused by pathogens such as virus, bacteria and malaria. A target DNA can be amplified within 30 min using the LAMP reaction, taking place at a steady temperature. The LAMP method uses four or six primers to bind eight regions of a target DNA and has a very high specificity. The devices used for conducting LAMP are usually simple since the LAMP method is an isothermal process. When LAMP is coupled with Reverse Transcription (RT), it allows direct detection of RNA in a sample. This greatly enhances the efficiency of diagnosis of RNA viruses in a sample. Recently, the rampant spread of COVID-19 demanded such a rapid, simple, and cost-effective Point of Care Test (PoCT) for the accurate diagnosis of this pandemic. Loop-mediated isothermal amplification (LAMP) assays are not only used for the detection of microbial pathogens, but there are various other applications such as detection of genetic mutations in food and various organisms. In this review, various implementations of RT-LAMP techniques would be discussed.
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Affiliation(s)
- Srishti Sen
- School of Bioscience, Engineering and Technology, VIT Bhopal University, Bhopal, Madhya Pradesh, India
| | - Priyanka Bhowmik
- Department of Biological Sciences, School of Life Science and Biotechnology, Adamas University, Kolkata, India
| | - Shubhangi Tiwari
- School of Bioscience, Engineering and Technology, VIT Bhopal University, Bhopal, Madhya Pradesh, India
| | - Yoav Peleg
- Structural Proteomics Unit (SPU), Life Sciences Core Facilities (LSCF), Weizmann Institute of Science, Rehovot, Israel
| | - Boudhayan Bandyopadhyay
- Department of Biotechnology, School of Life Science and Biotechnology, Adamas University, Kolkata, India.
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2
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Loop mediated isothermal amplification for detection of foodborne parasites: A journey from lab to lab-on-a-chip. Food Control 2022. [DOI: 10.1016/j.foodcont.2022.109251] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
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Haq F, Sharif S, Khurshid A, Ikram A, Shabbir I, Salman M, Ahad A, Suleman Rana M, Raja A, Badar N, Tashkandi H, Al Amri T, Azhar EI, Almuhayawi MS, Harakeh S, Faraz Arshad Malik M. Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)-based diagnosis: A potential alternative to quantitative real-time PCR based detection of the novel SARS-COV-2 virus. Saudi J Biol Sci 2020; 28:942-947. [PMID: 33424386 PMCID: PMC7785420 DOI: 10.1016/j.sjbs.2020.10.064] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Revised: 10/23/2020] [Accepted: 10/29/2020] [Indexed: 01/08/2023] Open
Abstract
The sudden outbreak of the novel Coronavirus infectious disease (COVID-19) resulted in significant challenges to global health systems. One of the primary challenges is rapid, reliable, and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) virus among the suspected COVID-19-infected individuals. At present, quantitative real-time PCR (qRT-PCR) is a widely used diagnostic method. However, it requires expensive instruments and expertise in the interpretation of results. These constraints reflect the significant need for the development of alternative diagnostic options. This study will validate the use and efficiency of the reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay as a potential alternative for the detection of COVID-19. For this purpose, a cohort of 297 suspected COVID-19 patients was tested using both the RT-LAMP assay and the conventional RT-PCR method. For the RT-LAMP assay, three genes (orf-1ab, N, and S) were identified as the target sites for the detection of COVID-19. Based on a comparative assessment, 117 out of 124 positive COVID-19 cases were observed using the RT-LAMP technique with an overall 91.45% sensitivity. Interestingly, where a consensus on 163 individuals free of SARS-Cov-2 was observed, RT-LAMP specificity was 90%. Based on these findings, the robustness of the technique, and the reduced dependency on expensive instrumentation, RT-LAMP-based COVID-19 detection is strongly recommended as a potential alternative assay.
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Affiliation(s)
- Farhan Haq
- Department of Biosciences, COMSATS University of Islamabad, Pakistan
| | | | | | - Aamer Ikram
- National Institute of Health, Islamabad, Pakistan
| | - Imran Shabbir
- Department of Biotechnology and Bioinformatics, International Islamic University, Islamabad, Pakistan
| | | | - Abdul Ahad
- National Institute of Health, Islamabad, Pakistan
| | | | - Aroosha Raja
- Department of Biosciences, COMSATS University of Islamabad, Pakistan
| | - Nazish Badar
- National Institute of Health, Islamabad, Pakistan
| | - Hanaa Tashkandi
- Department of Surgery, Faculty of Medicine (FM), King Abdulaziz University (KAU), Jeddah, Saudi Arabia
| | - Turki Al Amri
- Family and Community Medicine Department, Faculty of Medicine in Rabigh, KAU, Jeddah, Saudi Arabia
| | - Esam I Azhar
- Special Infectious Agents Unit, King Fahd Medical Research Center, Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, KAU, Saudi Arabia
| | | | - Steve Harakeh
- Special Infectious Agents Unit, King Fahd Medical Research Center, Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, KAU, Saudi Arabia.,Yousef Abdul Latif Jameel Scientific Chair of Prophetic Medicine Application, FM, KAU, Saudi Arabia
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Loop Mediated Isothermal Amplification: A Promising Tool for Screening Genetic Mutations. Mol Diagn Ther 2020; 23:723-733. [PMID: 31396882 DOI: 10.1007/s40291-019-00422-0] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Mutation screening is elemental for clinical diagnosis and in determining therapeutic strategies. Nucleic acid-based techniques are considered to be the most accurate tools in genetic diagnosis. One such technique is loop-mediated isothermal amplification (LAMP) assay, which has seen tremendous applications in recent years. The advantages of the assay lie in its rapidity, efficiency, sensitivity, and cost. It works in isothermal conditions and amplifies the target gene using DNA polymerases that have strand displacement activity. To date, the assay has been widely used in different fields of research, including pathogen detection, crop development, and disease diagnosis. However, despite the potential, its application in mutation screening has been minimal. This review highlights the LAMP assay and its variants that have been developed for screening single-nucleotide polymorphisms and gene translocations in cancer.
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Panno S, Matić S, Tiberini A, Caruso AG, Bella P, Torta L, Stassi R, Davino S. Loop Mediated Isothermal Amplification: Principles and Applications in Plant Virology. PLANTS (BASEL, SWITZERLAND) 2020; 9:E461. [PMID: 32268586 PMCID: PMC7238132 DOI: 10.3390/plants9040461] [Citation(s) in RCA: 99] [Impact Index Per Article: 19.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Revised: 04/02/2020] [Accepted: 04/02/2020] [Indexed: 01/14/2023]
Abstract
In the last decades, the evolution of molecular diagnosis methods has generated different advanced tools, like loop-mediated isothermal amplification (LAMP). Currently, it is a well-established technique, applied in different fields, such as the medicine, agriculture, and food industries, owing to its simplicity, specificity, rapidity, and low-cost efforts. LAMP is a nucleic acid amplification under isothermal conditions, which is highly compatible with point-of-care (POC) analysis and has the potential to improve the diagnosis in plant protection. The great advantages of LAMP have led to several upgrades in order to implement the technique. In this review, the authors provide an overview reporting in detail the different LAMP steps, focusing on designing and main characteristics of the primer set, different methods of result visualization, evolution and different application fields, reporting in detail LAMP application in plant virology, and the main advantages of the use of this technique.
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Affiliation(s)
- Stefano Panno
- Department of Agricultural, Food and Forest Sciences, University of Palermo, 90128 Palermo, Italy; (A.G.C.); (P.B.); (L.T.); (R.S.)
| | - Slavica Matić
- Department of Agricultural, Forestry and Food Sciences, University of Turin, 10095 Turin, Italy;
| | - Antonio Tiberini
- Council for Agricultural Research and Economics, Research Center for Plant Protection and Certification, 00156 Rome, Italy;
| | - Andrea Giovanni Caruso
- Department of Agricultural, Food and Forest Sciences, University of Palermo, 90128 Palermo, Italy; (A.G.C.); (P.B.); (L.T.); (R.S.)
| | - Patrizia Bella
- Department of Agricultural, Food and Forest Sciences, University of Palermo, 90128 Palermo, Italy; (A.G.C.); (P.B.); (L.T.); (R.S.)
| | - Livio Torta
- Department of Agricultural, Food and Forest Sciences, University of Palermo, 90128 Palermo, Italy; (A.G.C.); (P.B.); (L.T.); (R.S.)
| | - Raffaele Stassi
- Department of Agricultural, Food and Forest Sciences, University of Palermo, 90128 Palermo, Italy; (A.G.C.); (P.B.); (L.T.); (R.S.)
| | - Salvatore Davino
- Department of Agricultural, Food and Forest Sciences, University of Palermo, 90128 Palermo, Italy; (A.G.C.); (P.B.); (L.T.); (R.S.)
- Institute for Sustainable Plant Protection, National Research Council (IPSP-CNR), 10135 Turin, Italy
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Yu XF, Yang HJ, Lei L, Wang C, Huang J. CK19 mRNA in blood can predict non-sentinel lymph node metastasis in breast cancer. Oncotarget 2016; 7:30504-10. [PMID: 27105542 PMCID: PMC5058696 DOI: 10.18632/oncotarget.8851] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2015] [Accepted: 03/31/2016] [Indexed: 01/31/2023] Open
Abstract
Reverse-transcription polymerase chain reaction (RT-PCR) is used to detect CK19 mRNA in sentinel lymph node biopsy (SLNB) tissues from breast cancer patients. We examined whether CK19 mRNA in peripheral blood is predictive of non-sentinel lymph node (nSLN) metastasis. Breast cancer cases diagnosed with clinical stage cT1-3cN0 and registered in our medical biobank were identified retrospectively. This study then included 120 breast cancer cases treated at Zhejiang Cancer Hospital from Aug 2014 to Aug 2015, including 60 SLN-positive and 60 SLN-negative cases. CK19 mRNA levels in peripheral blood samples were assessed using RT-PCR prior to tumor removal. During surgery, if SLNB tissue showed evidence of metastasis, axillary lymph node dissection (ALND) was performed. No ALND was performed if SLNB and nSLN tissues were both negative for metastasis. CK19 expression was higher in nSLN-positive patients than in nSLN-negative patients (p < 0.05). Logistic regression indicated that lymphatic vessel invasion and CK19 levels were predictive of nSLN status (p < 0.05). The area under the ROC curve for CK19 was 0.878 (p < 0.05). We conclude that high CK19 levels in peripheral blood may independently predict nSLN metastasis in breast cancer patients.
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Affiliation(s)
- Xing-Fei Yu
- Department of Surgical Oncology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, P.R.China
- Department of Breast Tumor Surgery, Zhejiang Cancer Hospital, Banshan Bridge, Hangzhou, Zhejiang Province, 310022, P.R.China
| | - Hong-Jian Yang
- Department of Breast Tumor Surgery, Zhejiang Cancer Hospital, Banshan Bridge, Hangzhou, Zhejiang Province, 310022, P.R.China
| | - Lei Lei
- Department of Breast Medical Oncology, Zhejiang Cancer Hospital, Banshan Bridge, Hangzhou, Zhejiang Province, 310022, P.R.China
| | - Chen Wang
- Department of Breast Tumor Surgery, Zhejiang Cancer Hospital, Banshan Bridge, Hangzhou, Zhejiang Province, 310022, P.R.China
| | - Jian Huang
- Department of Surgical Oncology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310009, P.R.China
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Wu L, Wang B, Zhao M, Liu W, Zhang P, Shi Y, Xiong C, Wang P, Sun W, Chen S. Rapid Identification of Officinal Akebiae Caulis and Its Toxic Adulterant Aristolochiae Manshuriensis Caulis (Aristolochia manshuriensis) by Loop-Mediated Isothermal Amplification. FRONTIERS IN PLANT SCIENCE 2016; 7:887. [PMID: 27379153 PMCID: PMC4913086 DOI: 10.3389/fpls.2016.00887] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/17/2016] [Accepted: 06/06/2016] [Indexed: 05/07/2023]
Abstract
Mu-tong (Akebiae Caulis) is a traditional Chinese medicine commonly used as a diuretic and antiphlogistic. A common adulterant of Mu-tong is Guan-mu-tong (Aristolochiae Manshuriensis Caulis), which is derived from the stem of Aristolochia manshuriensis Komarov, and contains carcinogenic aristolochic acids. We used an alternative technique, loop-mediated isothermal amplification (LAMP), to differentiate Mu-tong from Guan-mu-tong because LAMP is quick, highly sensitive, and specific. We designed a set of four common primers (G-F3, G-B3, G-FIP, and G-BIP) and a loop primer (G-LB) for LAMP based on the internal transcribed spacer 2 sequence of Ar. manshuriensis. We successfully amplified the LAMP assays and visual detection occurred within 60 min at isothermal conditions of 65°C. The LAMP reaction exhibited a tenfold increase in detection (4.22 pg/μl DNA) over conventional polymerase chain reaction demonstrating that LAMP is a useful technique to detect Guan-mu-tong. We conclude that the LAMP technique is a potentially valuable safety control method for simple and efficient discrimination of Mu-tong from its adulterant Guan-mu-tong.
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Affiliation(s)
- Lan Wu
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical SciencesBeijing, China
- College of Pharmacy, Hubei University of Chinese MedicineWuhan, China
| | - Bo Wang
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical SciencesBeijing, China
| | - Mingming Zhao
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical SciencesBeijing, China
| | - Wei Liu
- Institute of Disease Control and Prevention, Academy of Military Medical SciencesBeijing, China
| | - Peng Zhang
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical SciencesBeijing, China
| | - Yuhua Shi
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical SciencesBeijing, China
| | - Chao Xiong
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical SciencesBeijing, China
| | - Ping Wang
- College of Pharmacy, Hubei University of Chinese MedicineWuhan, China
| | - Wei Sun
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical SciencesBeijing, China
- *Correspondence: Wei Sun, ; Shilin Chen,
| | - Shilin Chen
- Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical SciencesBeijing, China
- College of Pharmacy, Hubei University of Chinese MedicineWuhan, China
- *Correspondence: Wei Sun, ; Shilin Chen,
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Wang J, Lu W, Tang C, Liu Y, Sun J, Mu X, Zhang L, Dai B, Li X, Zhuo H, Jiang X. Label-Free Isolation and mRNA Detection of Circulating Tumor Cells from Patients with Metastatic Lung Cancer for Disease Diagnosis and Monitoring Therapeutic Efficacy. Anal Chem 2015; 87:11893-900. [PMID: 26531886 DOI: 10.1021/acs.analchem.5b03484] [Citation(s) in RCA: 87] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Affiliation(s)
- Jidong Wang
- Beijing Engineering Research Center for BioNanotechnology & CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190, China
| | - Wenjing Lu
- Beijing Engineering Research Center for BioNanotechnology & CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190, China
| | - Chuanhao Tang
- Affiliated Hospital of Academy of Military Medical Sciences (307 Hospital), No. 8 Dongdajie, Beijing, 100071, China
| | - Yi Liu
- Affiliated Hospital of Academy of Military Medical Sciences (307 Hospital), No. 8 Dongdajie, Beijing, 100071, China
| | - Jiashu Sun
- Beijing Engineering Research Center for BioNanotechnology & CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190, China
| | - Xuan Mu
- Peking
Union Medical College, Chinese Academy of Medical Sciences, Beijing, 100730, China
| | - Lin Zhang
- Peking
Union Medical College, Chinese Academy of Medical Sciences, Beijing, 100730, China
| | - Bo Dai
- Department
of Urology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China
| | - Xiaoyan Li
- Affiliated Hospital of Academy of Military Medical Sciences (307 Hospital), No. 8 Dongdajie, Beijing, 100071, China
| | - Hailong Zhuo
- Affiliated Hospital of Academy of Military Medical Sciences (307 Hospital), No. 8 Dongdajie, Beijing, 100071, China
| | - Xingyu Jiang
- Beijing Engineering Research Center for BioNanotechnology & CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190, China
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Wang K, Shao H, Pei Z, Hu G. Rapid detection of contagious ecthyma by loop-mediated isothermal amplification and epidemiology in Jilin Province China. J Vet Med Sci 2015; 78:125-8. [PMID: 26346652 PMCID: PMC4751130 DOI: 10.1292/jvms.15-0340] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
Abstract
The aim of this experiment was to develop a loop-mediated isothermal amplification (LAMP)
assay and to research the recent epidemiology of contagious ecthyma in Jilin Province,
China, using the assay. A LAMP assay targeting a highly conserved region of the F1L gene
was developed to detect contagious ecthyma virus (CEV). Three hundred and sixty-five cases
from 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 were
tested using the LAMP assay. The results showed that the sensitivity of the LAMP assay was
100 copies of the standard plasmid, which is 100-fold higher than the sensitivity of PCR.
No cross-reactivity was observed with capripoxvirus, fowlpox virus, foot-and-mouth disease
virus serotype O, foot-and-mouth disease virus serotype Asia I and bluetongue virus. The
average positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged
1–6 months. Our results demonstrated that CEV infection was very widespread in the flocks
of Jilin Province and that the LAMP assay allows for easy, rapid, accurate and sensitive
detection of CEV infection.
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Affiliation(s)
- Kai Wang
- College of Animal Science and Technology, Jilin Agricultural University, Xincheng Street No.2888, Changchun 130118, P.R. China
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Loop-mediated isothermal amplification for diagnosis of 18 World Organization for Animal Health (OIE) notifiable viral diseases of ruminants, swine and poultry. Anim Health Res Rev 2015; 16:89-106. [PMID: 25900363 DOI: 10.1017/s1466252315000018] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Loop-mediated isothermal amplification (LAMP) is a simple, powerful state-of-the-art gene amplification technique used for the rapid diagnosis and early detection of microbial diseases. Many LAMP assays have been developed and validated for important epizootic diseases of livestock. We review the LAMP assays that have been developed for the detection of 18 viruses deemed notifiable of ruminants, swine and poultry by the World Organization for Animal Health (OIE). LAMP provides a fast (the assay often takes less than an hour), low cost, highly sensitive, highly specific and less laborious alternative to detect infectious disease agents. The LAMP procedure can be completed under isothermal conditions so thermocyclers are not needed. The ease of use of the LAMP assay allows adaptability to field conditions and works well in developing countries with resource-limited laboratories. However, this technology is still underutilized in the field of veterinary diagnostics despite its huge capabilities.
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Sonoda H, Tani T. Clinical significance of molecular diagnosis for gastric cancer lymph node micrometastasis. World J Gastroenterol 2014; 20:13728-13733. [PMID: 25320510 PMCID: PMC4194556 DOI: 10.3748/wjg.v20.i38.13728] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/23/2013] [Revised: 03/14/2014] [Accepted: 06/26/2014] [Indexed: 02/06/2023] Open
Abstract
Advances in molecular diagnostic tools have allowed the identification of lymph node micrometastasis (LNM), including isolated tumor cells, in cancer patients. While immunohistochemistry and reverse transcription-polymerase chain reaction have been used to identify LNM in patients with gastric cancer, the clinical significance of this finding remains unclear. Recently, minimally invasive treatments, such as endoscopic submucosal dissection and laparoscopic surgery, are widely performed to help improve postsurgical quality of life (QOL). However, it is important to maintain the balance between QOL and curability when making treatments decision for patients with gastric cancer. If minimally invasive surgery based on accurate intraoperative LNM diagnosis was established, it could be performed safely. Therefore, we reviewed the clinical significance of LNM detected by molecular techniques as an important target for treatment decision making with gastric cancer patients.
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Hua X, Yin W, Shi H, Li M, Wang Y, Wang H, Ye Y, Kim HJ, Gee SJ, Wang M, Liu F, Hammock BD. Development of phage immuno-loop-mediated isothermal amplification assays for organophosphorus pesticides in agro-products. Anal Chem 2014; 86:8441-7. [PMID: 25135320 PMCID: PMC4139188 DOI: 10.1021/ac5020657] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
Two immuno-loop-mediated isothermal amplification assays (iLAMP) were developed by using a phage-borne peptide that was isolated from a cyclic eight-peptide phage library. One assay was used to screen eight organophosphorus (OP) pesticides with limits of detection (LOD) between 2 and 128 ng mL(-1). The iLAMP consisted of the competitive immuno-reaction coupled to the LAMP reaction for detection. This method provides positive results in the visual color of violet, while a negative response results in a sky blue color; therefore, the iLAMP allows one to rapidly detect analytes in yes or no fashion. We validated the iLAMP by detecting parathion-methyl, parathion, and fenitrothion in Chinese cabbage, apple, and greengrocery, and the detection results were consistent with the enzyme-linked immunosorbent assay (ELISA). In conclusion, the iLAMP is a simple, rapid, sensitive, and economical method for detecting OP pesticide residues in agro-products with no instrumental requirement.
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Affiliation(s)
- Xiude Hua
- College of Plant Protection (State & Local Joint Engineering Research Center of Green Pesticide Invention and Application), Nanjing Agricultural University , Nanjing 210095, P.R. China
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13
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Kanamori M, Kikuchi A, Watanabe M, Shibahara I, Saito R, Yamashita Y, Sonoda Y, Kumabe T, Kure S, Tominaga T. Rapid and sensitive intraoperative detection of mutations in the isocitrate dehydrogenase 1 and 2 genes during surgery for glioma. J Neurosurg 2014; 120:1288-97. [DOI: 10.3171/2014.3.jns131505] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Object
Intraoperative diagnosis is important in determining the strategies during surgery for glioma. Because the mutations in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes have diagnostic, prognostic, and predictive values, the authors assessed the feasibility and significance of a simplified method for the intraoperative detection of IDH1 and IDH2 gene mutations.
Methods
Rapid DNA extraction, amplification with conventional polymerase chain reaction (PCR) or co-amplification at lower denaturation temperature PCR (COLD-PCR), and fluorescence melting curve analysis with adjacent hybridization probes were performed for the intraoperative detection of IDH1 and IDH2 mutations in 18 cases of suspected nonneoplastic lesions and low- and high-grade gliomas and in 3 cases of radiation necrosis.
Results
DNA extraction for detection of the mutation took 60–65 minutes. The results of this assay showed complete correlation with that of Sanger sequencing. The sensitivity for detection of mutations in a background of wild-type genes was 12.5% and 2.5% in conventional PCR and COLD-PCR, respectively. The diagnosis of glioma was established in 3 of 5 cases in which definitive diagnosis was not obtained using frozen sections, and information was obtained for the discrimination of glioblastoma or glioblastoma with an oligodendroglioma component from anaplastic glioma or secondary glioblastoma. This assay also detected a small fraction of tumor cells with IDH1 mutation in radiation necrosis.
Conclusions
These methods provide important information for establishing the differential diagnosis between low-grade glioma and nonneoplastic lesions and the diagnosis for subtypes of high-grade glioma. Although tumor cells in radiation necrosis were detected with a high sensitivity, further investigation is necessary for clinical application in surgery for recurrent glioma.
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Affiliation(s)
| | - Atsuo Kikuchi
- 2Pediatrics, Tohoku University Graduate School of Medicine
| | - Mika Watanabe
- 3Department of Pathology, Tohoku University Hospital, Sendai
| | | | | | - Yoji Yamashita
- 4Department of Neurosurgery, Miyagi Cancer Center, Natori, Miyagi; and
| | | | - Toshihiro Kumabe
- 5Department of Neurosurgery, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan
| | - Shigeo Kure
- 2Pediatrics, Tohoku University Graduate School of Medicine
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Liu X, Tang J, Wang M, Ma Q, Wang Y. Visual detection and evaluation of latent and lytic gene expression during Epstein-Barr virus infection using one-step reverse transcription loop-mediated isothermal amplification. Int J Mol Sci 2013; 14:23922-40. [PMID: 24351866 PMCID: PMC3876086 DOI: 10.3390/ijms141223922] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2013] [Revised: 12/01/2013] [Accepted: 12/02/2013] [Indexed: 12/31/2022] Open
Abstract
Epstein-Barr virus (EBV)-associated disease exhibits distinct gene expression patterns characterized by the transcription of EBV nuclear antigen (EBNA) 1, EBNA2, latent membrane protein (LMP) 1, LMP2A, and BZLF1 (Zebra). A series of visual reverse transcript loop-mediated isothermal amplification (RT-LAMP) assays were performed to examine the expression of EBNA1, EBNA2, LMP1, LMP2A and BZLF1. The sensitivity of RT-LAMP for these transcripts was approximately equivalent to real-time RT-PCR (RT-qPCR), which was developed to quantify relative levels of EBV transcripts, and 10 to 100-fold more sensitive than conventional RT-PCR. Cross-reactions to other viruses were not observed upon examination of cell lines infected with herpes simplex viruses-1 and -2 (HSV-1 and -2), varicella zoster virus (VZV), human cytomegalovirus (HCMV) or Kaposi's sarcoma-associated herpesvirus. When applied to 146 specimens, RT-LAMP exhibited high clinical sensitivity and specificity, with an excellent agreement (κ > 0.92) compared to RT-qPCR. These assays are convenient for rapid early diagnosis and for surveillance of EBV-infected individuals by evaluating the EBV transcriptional profile, because the results can be visualized with the naked eye. These assays may be employed in further investigations because they can aid the design of improved therapeutic regimens and can be used specifically in resource-poor settings.
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Affiliation(s)
- Xiaoying Liu
- The State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, Hubei, China; E-Mails: (X.L.); (J.T.); (M.W.)
| | - Jingfeng Tang
- The State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, Hubei, China; E-Mails: (X.L.); (J.T.); (M.W.)
| | - Man Wang
- The State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, Hubei, China; E-Mails: (X.L.); (J.T.); (M.W.)
| | - Qiang Ma
- The State Key Laboratory of Respiratory Disease, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, Guangdong, China; E-Mail:
| | - Yefu Wang
- The State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, Hubei, China; E-Mails: (X.L.); (J.T.); (M.W.)
- Author to whom correspondence should be addressed; E-Mail: ; Tel.: +86-27-6875-4627; Fax: +86-27-6875-4592
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15
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Zhi X, Deng M, Yang H, Gao G, Wang K, Fu H, Zhang Y, Chen D, Cui D. A novel HBV genotypes detecting system combined with microfluidic chip, loop-mediated isothermal amplification and GMR sensors. Biosens Bioelectron 2013; 54:372-7. [PMID: 24292142 DOI: 10.1016/j.bios.2013.11.025] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2013] [Revised: 10/28/2013] [Accepted: 11/06/2013] [Indexed: 02/08/2023]
Abstract
Genotyping of hepatitis B virus (HBV) can be used for clinical effective therapeutic drug-selection. A novel microfluidic biochip for HBV genotyping has been fabricated, for the first time, integrating loop-mediated isothermal amplification (LAMP), line probes assay (LiPA) and giant magnetoresistive (GMR) sensors. Coupling LAMP with LiPA in microfluidic chip shortened reaction time substantially, and combining LAMP with GMR sensor enabled limit of detection to attain 10 copies mL(-1) target HBV DNA molecules in 1 h. Furthermore, the independent designed GMR sensors and microfluidic chip can decrease manufacturing cost and patient's test-cost, and facilitate GMR detector repeating use for signal detection. In addition, the detection system has a lower background signal owing to application of superparamagnetic nanoclusters. And it can be expected to use for multiple target molecules synchronous detection in microfluidic chip based on a characteristic of stationary reaction temperature of LAMP. In conclusion, the neoteric detecting system is well suitable for quick genotyping diagnosis of clinical HBV and other homothetic biomolecule detection in biological and medical fields.
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Affiliation(s)
- Xiao Zhi
- (a)National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, People's Republic of China
| | - Min Deng
- (a)National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, People's Republic of China
| | - Hao Yang
- (b)Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, No. 20 Dongda Street, Fengtai, Beijing 100071, P.R. China
| | - Guo Gao
- (b)Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, No. 20 Dongda Street, Fengtai, Beijing 100071, P.R. China
| | - Kan Wang
- (a)National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, People's Republic of China
| | - Hualin Fu
- (a)National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, People's Republic of China
| | - Yixia Zhang
- (a)National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, People's Republic of China
| | - Di Chen
- (a)National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, People's Republic of China
| | - Daxiang Cui
- (a)National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, People's Republic of China.
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16
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Feng C, Wang C, Lin X, Zhang Y, Lv J, Deng J, Yuan X, Mei L, Wu S. Development of a loop-mediated isothermal amplification method for detection of Perkinsus spp. in mollusks. DISEASES OF AQUATIC ORGANISMS 2013; 104:141-148. [PMID: 23709467 DOI: 10.3354/dao02591] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/02/2023]
Abstract
Perkinsus is a genus of unicellular protozoan parasite responsible for mass mortality of several commercially valuable mollusks. Surveillance and inspection of its epidemiology in the field calls for convenient and rapid detection methods. Here, a loop-mediated isothermal amplification (LAMP) assay was developed to detect the presence of Perkinsus spp. in mollusks. Specific LAMP primers were designed targeting the conserved internal transcribed spacer 2 (ITS-2) region of the rRNA gene of Perkinsus spp. Using ITS-2 recombinant plasmid as a template, we optimized the LAMP reaction system and conditions and then evaluated the analytical sensitivity and specificity of the assay. The LAMP assay was validated using clam samples collected from coastal areas in eastern China and oysters imported to China and compared with the traditional Ray's fluid thioglycollate culture method (RFTM). Our results showed that the LAMP detection method for Perkinsus was successful. The detection limit was 10 copies of plasmid DNA. Compared to the RFTM assay, the LAMP detection method was more sensitive (56 versus 52 positive out of 60 samples). P. olseni and P. marinus from infected hosts were successfully detected by this method. The LAMP method is rapid, sensitive, and specific for Perkinsus spp. detection, and could be used to screen for perkinsosis both on farms and at ports.
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Affiliation(s)
- Chunyan Feng
- Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100029, PR China
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17
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Almasi MA, Erfan Manesh M, Jafary H, Dehabadi SMH. Visual detection of Potato Leafroll virus by loop-mediated isothermal amplification of DNA with the GeneFinder™ dye. J Virol Methods 2013; 192:51-4. [PMID: 23680094 DOI: 10.1016/j.jviromet.2013.04.014] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2012] [Revised: 04/10/2013] [Accepted: 04/15/2013] [Indexed: 11/25/2022]
Abstract
The most common virus affecting potatoes in the field worldwide is Potato Leafroll virus (PLRV), belonging to the family Luteoviridae, genius Plerovirus. There are several molecular methods to detect PLRV including polymerase chain reaction (PCR), Multiplex AmpliDet RNA and double antibody sandwich ELISA (DAS-ELISA). But these techniques take a long time for 3h to two days, requiring sophisticated tools. The aim of this study was to reduce the time required to detect PLRV, using a newly designed loop-mediated isothermal amplification (LAMP) technique requiring only an ordinary water bath or thermoblock. PLRV RNA was extracted from overall 80 infected naturally potato leaves. A set of six novel primers for the LAMP reaction was designed according to the highly conserved sequence of the viral coat protein (CP) gene. LAMP was carried out under isothermal conditions, applying the Bst DNA polymerase enzyme; the LAMP products were detected visually using the GeneFinder™ florescence dye. A positive result using the GeneFinder™ dye was a color change from the original orange to green. Results confirmed LAMP with GeneFinder™ provides a rapid and safe assay for detection of PLRV. Since with other molecular methods, equipping laboratories with a thermocycler or expensive detector systems is unavoidable, this assay was found to be a simple, cost-effective molecular method that has the potential to replace other diagnostic methods in primary laboratories without the need for expensive equipment or specialized techniques. It can also be considered as a reliable alternative viral detection system in further investigations.
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Affiliation(s)
- Mohammad Amin Almasi
- Department of Agriculture and Plant Breeding, Faculty of Agriculture, Zanjan University, Zanjan, Iran
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18
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Li Q, Fang J, Liu X, Xi X, Li M, Gong Y, Zhang M. Loop-mediated isothermal amplification (LAMP) method for rapid detection of cry1Ab gene in transgenic rice (Oryza sativa L.). Eur Food Res Technol 2013. [DOI: 10.1007/s00217-013-1911-3] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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Yoneda A, Taniguchi K, Torashima Y, Susumu S, Kanetaka K, Kuroki T, Eguchi S. The detection of gastric cancer cells in intraoperative peritoneal lavage using the reverse transcription--loop-mediated isothermal amplification method. J Surg Res 2013; 187:e1-6. [PMID: 24360119 DOI: 10.1016/j.jss.2013.01.001] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2012] [Revised: 12/29/2012] [Accepted: 01/03/2013] [Indexed: 10/27/2022]
Abstract
INTRODUCTION To detect a small number of malignant cells, we used a highly sensitive detection system that measures the expression levels of cytokeratin (CK) 19 messenger RNA by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). MATERIALS AND METHODS We evaluated the clinical relevance of our novel diagnostic method with an RT-LAMP assay using CK19 as a target gene for the detection of free cancer cells in peritoneal lavage and assessed the clinical significance of the molecular diagnosis by survival analysis and frequency of recurrence, with a median follow-up period of 39 mo. We observed 52 patients with gastric cancer who underwent gastrectomy, bypass operation, and exploratory laparotomy. RESULTS Those 52 patients, who were subjected to both RT-LAMP and cytologic examination, were divided into the following three groups: (1) patients positive by cytology and RT-LAMP (CY+/LAMP+) (n = 9), (2) patients positive by LAMP and negative by cytology (CY-/LAMP+) (n = 12), and (3) patients negative by both cytology and LAMP (CY-/LAMP-) (n = 31). All patients with simultaneous peritoneal dissemination and positive cytology were positive on RT-LAMP. The results of RT-LAMP were statistically significant for recurrence by univariate analysis (P < 0.005). Cytology-positive cases had a very poor prognosis, and RT-LAMP-positive cases had a worse prognosis than RT-LAMP-negative cases. CONCLUSIONS Our findings suggest that CK19 RT-LAMP would be useful as an intraoperative diagnostic modality to detect patients with a high risk of recurrence even after clinically curative surgery, who thus require proper adjuvant therapy.
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Affiliation(s)
- Akira Yoneda
- Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
| | - Ken Taniguchi
- Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Yasuhiro Torashima
- Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Seiya Susumu
- Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Kengo Kanetaka
- Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Tamotsu Kuroki
- Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
| | - Susumu Eguchi
- Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
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20
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Loop-mediated isothermal amplification (LAMP) assay for rapid detection of Entamoeba histolytica in amoebic liver abscess. World J Microbiol Biotechnol 2012; 29:27-32. [PMID: 23054695 DOI: 10.1007/s11274-012-1154-7] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2012] [Accepted: 08/11/2012] [Indexed: 12/12/2022]
Abstract
Amoebic liver abscess (ALA) is the most common extra intestinal manifestation of invasive amoebiasis caused by Entamoeba histolytica. The lack of early and accurate diagnostic test to differentiate various causes of liver abscess necessitates more reliable laboratory diagnostic test. The present study was conducted to assess the applicability of Loop-Mediated Isothermal Amplification (LAMP) assay for detection of E. histolytica in ALA cases. Fifty patients (n = 50) with clinical suspicion of ALA were enrolled in the study. All the clinical samples were subjected to conventional PCR assay. LAMP assay was standardized and the results were compared with that of PCR assay. Out of fifty pus samples thirty-six (72 %, 36/50) were positive for E. histolytica with conventional PCR assay and forty-one (82 %, 41/50) were positive by LAMP assay. Thus, five additional positive cases, missed by conventional PCR were positive with LAMP assay. Apart from rapidity, operational simplicity of LAMP assay high specificity and sensitivity, one-step amplification, higher yield and immediate visual detection may serve as a better diagnostic tool for diagnosis of ALA.
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21
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Craw P, Balachandran W. Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. LAB ON A CHIP 2012; 12:2469-86. [PMID: 22592150 DOI: 10.1039/c2lc40100b] [Citation(s) in RCA: 508] [Impact Index Per Article: 39.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/07/2023]
Abstract
Nucleic Acid Testing (NAT) promises rapid, sensitive and specific diagnosis of infectious, inherited and genetic disease. The next generation of diagnostic devices will interrogate the genetic determinants of such conditions at the point-of-care, affording clinicians prompt reliable diagnosis from which to guide more effective treatment. The complex biochemical nature of clinical samples, the low abundance of nucleic acid targets in the majority of clinical samples and existing biosensor technology indicate that some form of nucleic acid amplification will be required to obtain clinically relevant sensitivities from the small samples used in point-of-care testing (POCT). This publication provides an overview and thorough review of existing technologies for nucleic acid amplification. The different methods are compared and their suitability for POCT adaptation are discussed. Current commercial products employing isothermal amplification strategies are also investigated. In conclusion we identify the factors impeding the integration of the methods discussed in fully automated, sample-to-answer POCT devices.
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Affiliation(s)
- Pascal Craw
- Department of Electronic & Computer Engineering, School of Engineering & Design, Brunel University, London, UK.
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22
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Fu S, Qu G, Guo S, Ma L, Zhang N, Zhang S, Gao S, Shen Z. Applications of loop-mediated isothermal DNA amplification. Appl Biochem Biotechnol 2010; 163:845-50. [PMID: 20844984 DOI: 10.1007/s12010-010-9088-8] [Citation(s) in RCA: 102] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2010] [Accepted: 09/07/2010] [Indexed: 10/19/2022]
Abstract
During the last 10 years, with the development of loop-mediated isothermal amplification (LAMP) method, it has been widely applied in nucleic acid analysis because of its simplicity, rapidity, high efficiency, and outstanding specificity. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. Expensive equipment are not necessary to acquire a high level of precision, and there are fewer preparation steps compared to conventional PCR and real-time PCR assays. This paper briefly summarized the applications of LAMP method in pathogenic microorganisms, genetically modified ingredients, tumor detection, and embryo sex identification.
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Affiliation(s)
- Shijun Fu
- Key Laboratory of Preventive Veterinary Medicine and Animal Biotechnology, Binzhou Animal Husbandry and Veterinary Research Academy, Binzhou 256600, People's Republic of China.
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23
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Maeda J, Inoue M, Nakabayashi K, Otomo Y, Shintani Y, Ohta M, Okumura M, Matsuura N. Rapid diagnosis of lymph node metastasis in lung cancer with loop-mediated isothermal amplification assay using carcinoembryonic antigen–mRNA. Lung Cancer 2009; 65:324-7. [DOI: 10.1016/j.lungcan.2008.12.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2008] [Revised: 11/20/2008] [Accepted: 12/01/2008] [Indexed: 11/25/2022]
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Yanagita S, Natsugoe S, Uenosono Y, Arigami T, Arima H, Kozono T, Funasako Y, Ehi K, Nakajo A, Ishigami S, Aikou T. Detection of micrometastases in sentinel node navigation surgery for gastric cancer. Surg Oncol 2008; 17:203-210. [PMID: 18539025 DOI: 10.1016/j.suronc.2008.04.008] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Although lymph node metastasis is one of the important prognostic factors for patients with gastric cancer, the clinical significance of micrometastasis remains controversial. In the 6th edition of the TMN classification, micrometastases were classified as micrometastasis (MM) and isolated tumor cells (ITC) according to its greatest dimension. The accurate diagnosis of micrometastases is required when considering less invasive surgery, especially in early stage of gastric cancer. Since generating useful information about micrometastases by conventional RT-PCR is time-consuming, this procedure is not useful for rapid diagnosis during surgery. Recently some new methods of genetic diagnosis have reduced the amount of time required to obtain information about micrometastases in lymph nodes to 30-40 min. Such methodology can be clinically applied during less invasive surgery. The sentinel node (SN) concept has recently been applied to gastric cancer and SN navigation surgery (SNNS) is ideal for reduction of lymphadenectomy in patients with early gastric cancer. However, we should think about some conditions to establish SN concept for gastric cancer: the particle size of radioisotope, relationship between metastatic area and RI uptake, and the diagnosis of micrometastases by various method such as histological examination, immunostaining and RT-PCR. Here, we described the current status of MM and ITC in the lymph nodes and the SN concept in gastric cancer.
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Affiliation(s)
- Shigehiro Yanagita
- Department of Surgical Oncology, Field of Oncology, Course of Advanced Therapeutics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan.
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25
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Visser M, Jiwa M, Horstman A, Brink AATP, Pol RP, van Diest P, Snijders PJF, Meijer CJLM. Intra-operative rapid diagnostic method based on CK19 mRNA expression for the detection of lymph node metastases in breast cancer. Int J Cancer 2008; 122:2562-7. [PMID: 18324628 PMCID: PMC2658031 DOI: 10.1002/ijc.23451] [Citation(s) in RCA: 133] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Staging by sentinel node (SN) biopsy is the standard procedure for clinically node-negative breast cancer patients. Intra-operative analysis of the SN allows immediate axillary lymph node (ALN) dissection in SN positive patients, but a quick, reliable and reproducible method is lacking. We tested the suitability of a quantitative cytokeratin 19 (CK19) mRNA one step nucleic acid amplification (OSNA#) technique (OSNA-CK19) for intra-operative SN analysis. OSNA-CK19 involves a short manual sample preparation step and subsequent fully automated amplification of CK19 mRNA based on reverse transcription loop-mediated isothermal amplification, with results available within 30–40 min. OSNA-CK19 was compared to histological staining (Hematoxylin&Eosin and CAM5.2 and CK19 immunostaining) of 346 frozen ALNs from 32 breast cancer patients, using half of the lymph node for each method. 267 samples were negative and 61 positive by both methods. Three samples were histology positive and OSNA-CK19 negative. Fifteen samples were histology negative and OSNA-CK19 positive, 11 of which had copy numbers close to the cut-off level of OSNA-CK19. Seven of these 15 samples were RT-PCR positive for epithelial markers and/or showed CK19 protein expression by Western blot suggesting the presence of tumor deposits in the lymph node part investigated by OSNA-CK19. Concordance with histology was 94.8%, and 96.8% after exclusion of the latter 7 discordant cases. Sensitivity was 95.3% and specificity was 94.7% before and 97.1% after discordant case investigation. Our results indicate that OSNA-CK19 can potentially be useful in an intra-operative clinical setting to detect SN tumor involvement in breast cancer patients.
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Affiliation(s)
- Mike Visser
- Department of Pathology, VU Medical Center Amsterdam, The Netherlands
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26
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Fujiwara Y, Doki Y, Taniguchi H, Sohma I, Takiguchi S, Miyata H, Yamasaki M, Monden M. Genetic detection of free cancer cells in the peritoneal cavity of the patient with gastric cancer: present status and future perspectives. Gastric Cancer 2008; 10:197-204. [PMID: 18095074 DOI: 10.1007/s10120-007-0436-5] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/24/2007] [Accepted: 08/28/2007] [Indexed: 02/07/2023]
Abstract
The purpose of this review is to examine the current status and future perspectives of the molecular analysis of peritoneal lavage fluid in patients with gastric cancer. During the past 10 years, the polymerase chain reaction (PCR) has been applied for the molecular detection of free cancer cells in the abdominal cavity of patients with gastric cancer, and its clinical significance in establishing the presence of peritoneal dissemination has been assessed by several groups especially in Japan. The majority of these studies have confirmed the predictive value of the molecular detection of peritoneal metastasis and recurrence using peritoneal lavage fluid. Based on these findings, since April 2006, the genetic diagnosis of body fluids has been included in the Japanese Government public health insurance program for patients with solid tumors. However, there are still many obstacles to overcome before the genetic diagnosis of micrometastasis can be considered a routine laboratory assay. Here we review the importance of the molecular detection of cancer cells in the abdominal cavity, and the molecular techniques used for such diagnosis; we also provide some clinical examples to illustrate the value of molecular diagnosis.
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Affiliation(s)
- Yoshiyuki Fujiwara
- Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka (E-2), Suita, 565-0871, Japan
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27
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Ikeda S, Takabe K, Inagaki M, Funakoshi N, Suzuki K. Detection of gene point mutation in paraffin sections using in situ loop-mediated isothermal amplification. Pathol Int 2007; 57:594-9. [PMID: 17685931 DOI: 10.1111/j.1440-1827.2007.02144.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
For pathological diagnoses, visualization of genetic status using routine tissue sections is important to determine the relationships between histopathological findings and genetic alterations. Loop-mediated isothermal amplification (LAMP) has been reported to have high levels of specificity and amplification efficiency. An in situ LAMP method was used, along with an amplification refractory mutation system (ARMS) to directly detect a specific point mutation, L858R, which is a mutation of epidermal growth factor receptor (EGFR), useful for the prediction of the effects of the anti-lung cancer drug gefitinib. The investigation was done using two types of cultured cells as well as paraffin-sectioned specimens collected from 26 cases of surgically resected lung cancer. Twelve of the specimens had an L858R mutation and in situ LAMP showed reactions in the nuclei of all cancer cells present in those. Such reactions were also shown on in situ LAMP in three of the remaining 14 cases that were without the L858R mutation. In addition, a few cases showed responses in the nuclei of bronchial epithelium cells located in non-cancerous areas in the vicinity of a positive tumor, which suggested that the mutation had already occurred in the tumorigenic early stage. It is concluded that the present method is useful for pathological and genetic diagnoses.
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Affiliation(s)
- Satoshi Ikeda
- Department of Pathology, Tsuchiura Kyodo General Hospital, Ibaraki, Japan.
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28
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Katsuragi K, Yashiro M, Sawada T, Osaka H, Ohira M, Hirakawa K. Prognostic impact of PCR-based identification of isolated tumour cells in the peritoneal lavage fluid of gastric cancer patients who underwent a curative R0 resection. Br J Cancer 2007; 97:550-6. [PMID: 17667927 PMCID: PMC2360343 DOI: 10.1038/sj.bjc.6603909] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Identification of cancer cells in the peritoneal cavity could influence therapy and outcome of gastric carcinoma patients. The objective of this study was to evaluate the clinical impact of the real-time quantitative polymerase chain reaction-(PCR) based identification of isolated tumour cells in the peritoneal lavage fluid of gastric carcinoma. The peritoneal lavage fluid of 116 patients with gastric cancer was sampled at laparotomy. After RNA extraction and reverse transcription, real-time quantitative PCR was performed using the primers and probes for carcinoembryonic antigen (CEA) and cytokeratin-20 (CK20). When either the CEA mRNA or CK20 mRNA level of the sample was over the cutoff value, the sample was determined to be PCR-positive. Forty-six (40%) of the 116 patients were PCR-positive and 30 (65%) of the 46 PCR-positive patients died as a result of recurrent peritoneal dissemination. The prognosis of the 46 PCR-positive patients was significantly (P<0.001) worse than that of 70 PCR-negative patients. Furthermore, in 80 of the cases with a curative R0 resection, 15 of the patients with PCR-positive findings had a significantly (P<0.001) poorer prognosis than the 65 PCR-negative patients. The prognosis of the PCR-positive patients was significantly poorer than that of the PCR-negative patients in the T3 (P<0.0001) and T4 (P=0.048) subgroups. In a multivariate analysis of the 80 cases with a curative R0 resection, the real-time quantitative RT–PCR (CEA and/or CK20) levels indicated that they were independent prognostic factors. The real-time quantitative RT–PCR analysis of the CEA and/or CK20 transcripts in the peritoneal lavage fluid is useful for predicting the peritoneal recurrence in patients who are undergoing a curative resection for gastric cancer.
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Affiliation(s)
- K Katsuragi
- Department of Surgical Oncology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
| | - M Yashiro
- Department of Surgical Oncology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
- E-mail:
| | - T Sawada
- Department of Surgical Oncology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
| | - H Osaka
- Department of Surgical Oncology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
| | - M Ohira
- Department of Surgical Oncology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
| | - K Hirakawa
- Department of Surgical Oncology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
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