应用聚合酶链反应技术检测沙门氏菌

Detection of Salmonella by Polymerase Chain Reaction

利用聚合酶链反应（ＰＣＲ）检测沙门氏菌，使用了２对引物ＨＩ和ｐｈｏＰ。ＨＩ引物对各长２０ＮＴ，根据鞭毛Ｉ相抗原基因自行设计，扩增序列长２６９ｂｐ；ｐｈｏＰ引物对各长２１ＮＴ，根据ｐｈｏＰ／ｐｈｏＱ基因设计，扩增片段长２９９ｂｐ。ＰＣＲ采用５０μＬ反应体系。ｄＮＴＰＳ各１００μｍｏｌ／Ｌ，引物各１μｍｏｌ／Ｌ，Ｍｇ２＋２．５ｍｍｏｌ／Ｌ，酶２Ｕ。三温段ＰＣＲ循环条件为：９７℃预变性７ｍｉｎ；９４℃变性６０ｓ、５５℃复性４０ｓ、７２℃延伸６０ｓ，３５个循环；７２℃延伸７ｍｉｎ。检测８株标准阳性菌，结果都出现了ＨＩ引物和ｐｈｏＰ引物特异带，标准阴性菌３株只出现ｐｈｏＰ特异带，说明ＨＩ引物特异性很强。

Two sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. HI primers targeted a 269 bp region of HI gene specific to Salmonella species; While phoP primers targeted a 299 bp region of phoP/phoQ loci of coliformpathogenic bacteria. The standard PCR was typically done in a 50 μL Volume. It contained 2.5 mmol/L MgCl 2, 1 μmol/L of each primer, 200 μmol/L of each deoxynucleotide trisphate, 10 6 10 7 copies of templates DNA, 50 mmol/L KCl, 10 mmol/L Tris HCl, 10 mg/L gelatin and 2 units of Taq polymerase. The amplications were performed in two ways: A: Condition:heat denaturation at 97℃ for 7 min and then an additional 35 cycle with heat denaturation at 94℃ for 1 min, primer annealing at 55℃ 40 s, and DNA extension at 72℃ for 1 min. After the last cycle, samples were maintained at 72℃ 7 min. This kind of amplification was performed on a DNA thermal cycler. B: Condition:heat denaturation at 97℃ for 7 min and an additional 35 cycles with heat denatruation at 94℃ for 1 min, primer annealing and DNA extension at 60℃ for 1 min. After the last cycle, samples were maintained at 72℃ for 7 min. This kind of amplification was done by mannual operation between two temperature control stations. Eleven known bacterial strains, including 8 Salmonella species, 2 shigella species, 1 E.coli strain were analyzed. 269 bp band could be seen only in Salmonella species, and 299 bp band could be seen in all of them indicating the 269 bp region of HI gene may be specific to Salmonella species.