伤寒和甲、乙、丙型副伤寒沙门菌多重PCR及荧光PCR分型方法的建立与评价

The multiplex PCR and the real-time PCR assay for rapid detection of S. paratyphi A, S. paratyphi B, S. paratyphi C and S typhi

目的建立伤寒和甲、乙、丙型副伤寒沙门菌的快速和特异的检测方法,用于沙门菌属内的分型鉴定。方法根据GenBank公布的伤寒和甲、乙、丙型副伤寒沙门菌的保守序列设计引物和改良分子信标探针,建立多重PCR检测方法和实时荧光PCR检测方法。结果采用所建立的多重PCR方法可分别检测到伤寒的3条特异性条带、甲型副伤寒沙门菌的2条特异性条带和丙型副伤寒沙门菌的1条特异性条带,但是乙型副伤寒沙门菌未出现特异性条带。建立的实时荧光PCR方法可以快速、特异、灵敏地检测出伤寒和甲、乙、丙型副伤寒沙门菌,纯DNA和菌液的最低检出限分别可达10 fg/reaction和20 CFU/reaction;对77株细菌的检测正确率达100%。结论建立的实时荧光PCR检测方法比多重PCR方法更能快速、特异、灵敏地检测伤寒和甲、乙、丙型副伤寒沙门菌。

Objective To develop rapid PCR assay for the simultaneous detection of S. paratyphi A, S. paratyphi B, S. paratyphi C and S typhi. The method would be applied to the rapid diagnosis and subtype of Salmonella. Methods Based on the sequences of published on GeneBank, primers and modified molecular beacon probes were designed. Then the multiplex PCR and the real-time PCR assay for the simultaneous detection of S. paratyphi A, S. paratyphi B, S. paratyphi C and S typhi was developed. Results When using the multiplex PCR assay, there are there specific stripes of S typhi, two specific stripes of S. paratyphi A and one specific stripe of S. paratyphi C can be detected. The real-time PCR assay can rapidly detect S. paratyphi A, S. paratyphi B, S. paratyphi C and S typhi. The real-time PCR assay based on modified molecular beacons, the sensitivity achieved was 10 fg/reaction or 20 CUF/reaction. There was no cross-reaction with other bacteria as control. The assay was tested against 77 strains, then no false signals were observed and the specific was 100%. Conclusions The real-time PCR assay was rapid,sensitive and specific. It could be applied to the rapid diagnosis of S. paratyphi A, S. paratyphi B, S. paratyphi C and S typhi.