Brief Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. May 28, 2013; 19(20): 3090-3095
Published online May 28, 2013. doi: 10.3748/wjg.v19.i20.3090
Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection
Mao-Jun Zhang, Bo Qiao, Xue-Bin Xu, Jian-Zhong Zhang
Mao-Jun Zhang, Bo Qiao, Jian-Zhong Zhang, State Key Laboratory for Infectious Disease Prevention and Control, and National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
Xue-Bin Xu, Shanghai Municipal Center for Disease Control and Prevention, Shanghai 200336, China
Author contributions: Zhang MJ designed the research, analyzed the data and wrote the paper; Qiao B performed the research and analyzed the data; Xu XB collected the clinical samples and analyzed the data; Zhang JZ analyzed the data.
Supported by The General Program of National Natural Science Foundation of China, No. 81271789; and the Major State Basic Research Development Program, No. 2013CB127204
Correspondence to: Dr. Mao-Jun Zhang, State Key Laboratory for Infectious Disease Prevention and Control, and National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, PO Box 5, Changping District, Beijing 102206, China. zhangmaojun@icdc.cn
Telephone: +86-10-58900755 Fax: +86-10-58900700
Received: January 15, 2013
Revised: March 7, 2013
Accepted: April 9, 2013
Published online: May 28, 2013
Processing time: 133 Days and 11.7 Hours
Abstract

AIM: To develop a real-time polymerase chain reaction (PCR) method to detect and quantify Campylobacter jejuni (C. jejuni) from stool specimens.

METHODS: Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C. jejuni. The specificity of the primers and probe were tested against a set of Campylobacter spp. and other enteric pathogens. The optimal PCR conditions were determined by testing a series of conditions with standard a C. jejuni template. The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen. Two hundred and forty-two specimens were analyzed for the presence of C. jejuni by direct bacterial culture and real-time PCR.

RESULTS: The optimal PCR system was determined using reference DNA templates, 1 × uracil-DNA glycosylase, 3.5 mmol/L MgCl2, 1.25 U platinum Taq polymerase, 0.4 mmol/L PCR nucleotide mix, 0.48 μmol/L of each primer, 0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL. The PCR reaction was carried as follows: 95 °C for 4 min, followed by 45 cycles of 10 s at 95 °C and 30 s at 59 °C. The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 103 CFU/g using DNA from stool specimens. Twenty (8.3%, 20/242) C. jejuni strains were isolated from bacterial culture, while 41 (16.9%, 41/242) samples were found to be positive by real-time PCR. DNA sequencing of the PCR product indicated the presence of C. jejuni in the specimen. One mixed infection of C. jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.

CONCLUSION: The sensitivity of detection of C. jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.

Keywords: Campylobacter jejuni; Real time polymerase chain reaction; Application

Core tip: In the present study, we developed an effective real-time polymerase chain reaction method based on the specific DNA sequence of the hipO gene in Campylobacter jejuni (C. jejuni). The detection limit of this assay is 4.3 CFU/mL. A study of 242 clinical stool specimens from diarrheal patients indicated that this method is more sensitive than direct bacterial culture for the identification of C. jejuni from stool specimens.