Shigella LAMP Detection Method Based on ipaH Gene: Establishment and Application

doi:10.11924/j.issn.1000-6850.casb2020-0084

Abstract

Abstract:

To establish a LAMP detection method which is rapid, specific and sensitive for Shigella, the sequence of invasion plasmid antigen H gene (invasion plasmid, ipaH) of Shigella was used as target sequence to design a set of specific primers, the LAMP method was established after condition optimization, specificity and sensitivity verification, and applied to detect clinical samples. The results showed that the amplification products could be obtained within 1 h at 62℃. The standard Shigella strains showed the positive result, while Salmonella, Escherichia coli, Staphylococcus aureus, Bacillus subtilis and the negative control showed the negative result. The detection limit of the LAMP method for Shigella was 120 fg/μL. 20 suspected strains and 38 fecal samples were tested by the established method. The results showed that 20 suspected bacteria and 27 fecal samples were positive, and the positive rate was 100% and 71.05%, respectively, indicating that this LAMP assay was a specific and sensitive method, and it had advantages and application prospects in the detection and identification of Shigella in clinical samples such as feces.

Key words: loop-mediated isothermal amplification assay, Shigella, ipaH, detect

CLC Number:

S858.2

Wen Ying, Liu Yongsheng. Shigella LAMP Detection Method Based on ipaH Gene: Establishment and Application[J]. Chinese Agricultural Science Bulletin, 2021, 37(5): 104-110.

Figures/Tables 10

References 27

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基因	引物名称	引物序列 (5’-3’)
ipaH	LF	GAGTTTTTCCAGCCATGCAG
	LB	TGCCACTGAGAGCTGTGAG
	FIP	CTGTCGAAGCTCCGCAGAGGGGATTCCGTGAACAGGTCG
	BIP	AGTCTTTCGCTGCTGCTGCTGATCCGGAGATTGTTCCATGTG
	F3	ATACCGTCTCTGCACGCA
	B3	GCCTTCTGATGCCTGATGG

基因	引物名称	引物序列 (5’-3’)
ipaH	LF	GAGTTTTTCCAGCCATGCAG
	LB	TGCCACTGAGAGCTGTGAG
	FIP	CTGTCGAAGCTCCGCAGAGGGGATTCCGTGAACAGGTCG
	BIP	AGTCTTTCGCTGCTGCTGCTGATCCGGAGATTGTTCCATGTG
	F3	ATACCGTCTCTGCACGCA
	B3	GCCTTCTGATGCCTGATGG

试剂	用量
2×反应缓冲液(RM)	12.5 μL
F3	1.0 μL
B3	1.0 μL
Loop-F	1.0 μL
Loop-B	1.0 μL
FIP	1.0 μL
BIP	1.0 μL
DNA模板	2.0 μL
BstDNA聚合酶	1.0 μL
去离子水(DW)	3.5 μL
合计	25.0 μL

试剂	用量
2×反应缓冲液(RM)	12.5 μL
F3	1.0 μL
B3	1.0 μL
Loop-F	1.0 μL
Loop-B	1.0 μL
FIP	1.0 μL
BIP	1.0 μL
DNA模板	2.0 μL
BstDNA聚合酶	1.0 μL
去离子水(DW)	3.5 μL
合计	25.0 μL