Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

Table 1 Primer sequences for the Helicobacter pylori 23SrRNA gene

Gene name	Primer sequence, 5’-3’	Product length (bp)
External-F	GCGTTGAATTGAAGCCCGAGTAAAC	505
External-R	CCGACTTTCGTCTCTGCTTGA
WT2142-F	TCCTACCCGCGGCAAGACGGA	294
MUT2142-F	TCCTACCCGCGGCAAGACGGG
WT2143-F	CCTACCCGCGGCAAGACGGAA
MUT2143-F	CCTACCCGCGGCAAGACGGAG
WT2144-F	CCTACCCGCGGCAAGACGGAAA
MUT2144-F	CCTACCCGCGGCAAGACGGAAG
Inner-R	GCCATTACACTCAACTTGCGATTTC

The underlined primer is designed to match the single nucleotide polymorphism site. WT: Wild-type; MUT: Mutation; F: Forward primer; R: Reverse primer.

Table 2 Results for the 23SrRNA point mutation in 30 gastric mucosa and saliva samples, as detected by ASP-PCR and nested-ASP-PCR

	H. pylori-positive detection rate	WT	Total mutation, A-to-G	2142 G	2143 G	2144 G
Gastric mucosa
ASP-PCR	19	8	11	10	10	10
Ratio	63%	42.11%	57.89	52.63%	52.63%	52.63%
	(19/30)	(8/30)	(11/19)	(10/19)	(10/19)	(10/19)
Nested-ASP-PCR	27	16	11	11	10	11
Ratio	90%	59.26%	40.74%	40.74%	37.04%	40.74%
	(27/30)	(16/30)	(11/19)	(11/19)	(10/19)	(10/19)
Saliva
ASP-PCR	17	7	10	10	4	0
Ratio	56.67%	41.18%	58.82%	58.82%	23.53%	0
	(17/30)	(7/30)	(10/17)	(10/17)	(4/17)
Nested-ASP-PCR	25	9	16	16	13	9
Ratio	83.33%	36%	64%	64%	52%	36%
	(25/30)	(9/30)	(16/25)	(16/25)	(13/25)	(9/25)

ASP-PCR: Allele-specific primer-polymerase chain reaction.

Table 3 Results for the 23SrRNA point mutation in gastric mucosa and saliva samples of 99 RUT-positive clinical cases detected by nested-ASP-PCR

	RUT-positive	H. pylori-positive detection rate	WT	Total mutation, A-to-G	2142 G	2143 G	2144 G
Gastric mucosa	99	87	30	57	50	52	44
ratio		87.88%	34.48%	65.52%	57.47%	59.77%	50.57%
		(87/99)	(30/87)	(57/87)	(50/87)	(52/87)	(44/87)
Saliva	99	67	22	45	35	39	23
ratio		67.68%	32.84%	67.16%	52.24%	58.21%	34.34%
		(67/99)	(22/67)	(45/67)	(35/67)	(39/67)	(22/67)

H. pylori: Helicobacter pylori; ASP-PCR: Allele-specific primer-polymerase chain reaction.

Table 4 Results for Helicobacter pylori detection and clarithromycin resistance in gastric mucosa by different testing methods

	RUT-positive	Nested-ASP-PCR detection	Nested-ASP-PCR mutation	Bacterial culture H. pylori-positive	Drug-resistant strains
Cases	99	87	57	50	26
Ratio		87.88% (87/99)	65.52% (57/87)	57.47% (50/87)	52% (26/50)

H. pylori: Helicobacter pylori; ASP-PCR: Allele-specific primer-polymerase chain reaction.