应用PCR技术检测沙门氏菌

Application of Polymerase Chain Reaction for Detection of Salmonella

参照文献报道的根据沙门氏菌组氨酸转运操纵子基因片段所设计的引物序列，合成了１对长２５ｂｐ的引物。对沙门氏菌属Ａ～Ｆ各群中共１６株沙门氏标准菌株和其他１２株非沙门氏菌标准菌株进行ＤＮＡ抽提扩增，结果沙门氏菌ＰＣＲ产物都出现４９５ｂｐ的特异性ＤＮＡ扩增带，而非沙门氏菌均未出现扩增条带，证明这对引物具有沙门氏菌属特异性。选用ＨｉｎｄⅢ内切酶对ＰＣＲ产物直接进行酶切，紫外灯下可见２条电泳带，其长度（大片段长度为３１４ｂｐ，小片段为１８０ｂｐ）与目的基因序列相符合。用低融点琼脂糖对ＰＣＲ产物电泳回收、纯化，采用随机引物法以α－３２Ｐ－ｄＣＴＰ标记探针，分别与沙门氏菌和非沙门氏菌靶ＤＮＡ进行Ｄｏｔｂｌｏｔ和Ｓｏｕｔｈｅｒｎｂｌｏｔ，杂交后用普通Ｘ光胶片自显影，结果探针只与沙门氏菌ＤＮＡ杂交，并出现很强的杂交信号，而与非沙门氏菌不杂交，证实扩增产物是目的基因片段。以稀释的核酸分子量标准λＤＮＡ／ＨｉｎｄⅢ酶切片段作对照，将抽提的沙门氏菌ＤＮＡ作系列稀释，测定该ＰＣＲ体系的敏感度，结果表明，此ＰＣＲ体系能检出３０ｐｇ以上的细菌ＤＮＡ。采用上述ＰＣＲ技术，对沙门氏菌人工感染发病、自然发病小鼠、雏鸡的血液、脏器、粪便进行检测，?

Based on the primer sequence reported in literature, a pair of 25 oligonucleotide primers for the polymerase chain reaction (PCR) that amplify a 495 nucleotide sequence within the Salmonella histidine transport operon was synthesized. DNA extracted from 16 strains of Salmonella belonging to A F groups was amplified and DNA from 12 strains of non Salmonella was used for control. Products of the PCR were electrophoresed on 1% agl gel containing ethidium bromide, visualized with an ultraviolet transillumintor and photographed. All Salmonella revealed positive result indicated by the presence of a 495 bp band on the gel, while non Salmonella bacteria gave negative results demonstrating that the primers should be specific for all members of the Salmonella genus tested. Hind Ⅲ restriction endonuclease was used to cleave the PCR products. Two fragments were seen under an ultraviolet transilluminator, the big one being 314 bp and the small one 180 bp in size, which would be expected of the target sequence. One of 495 bp PCR products excised from low melting point gel and purified was labelled with α 32 P dCTP by random primer labelling and utilized as a specific probe. The probe was used to hybridize with chromosomal DNA of Salmonella and non Samonella bacteria for dot blotting and southern blotting. Strong hybridization signals occurred only with Salmonella DNA. The PCR would be able to detect as little as 30 pg Samonella DNA as determined by amplification of serially diluted Samonella DNA. A 495 bp segment could be amplified from extracted Salmonella DNA in organ, blood and fecal samples from chickens and white mice infected with Salmonella. The whole process of DNA extraction amplification and electrophoresis could be finished within 8 10 hours. The results indicated that the PCR method is a specific, sensitive and rapid one for identification of Salmonella.