Optimisation of the PCR-invA primers for the detection of Salmonella in drinking and surface waters following a pre-cultivation step
Optimisation of the PCR-invA primers for the detection of Salmonella in drinking and surface waters following a pre-cultivation step
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预培养步骤后用于检测饮用水和地表水中沙门氏菌的 PCR-invA 引物的优化
DOI：
10.4314/wsa.v33i2.49060
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发表时间：
2009
期刊：
影响因子：
1.5
通讯作者：
M. Sibara
中科院分区：
文献类型：
--
作者：
K.L.M. Moganedi;E. Goyvaerts;S. Venter;M. Sibara
一种基于聚合酶链反应（PCR）的方法检测沙门氏菌的水样进行了优化和评价的速度，特异性和灵敏度。Mg 2+和引物浓度以及循环参数的优化将PCR的灵敏度和检测限提高到2.6 x 104 cfu/m2。6小时的非选择性预富集步骤进一步将检测限提高到26 cfu/m2。在测试的14种不同沙门氏菌菌株中，只有两种，亚利桑那沙门氏菌和鸡白痢沙门氏菌，用与invA基因的保守区域同源的引物没有得到阳性扩增结果。当环境和饮用沃茨进行评估，一个非选择性的预富集步骤，包括增加PCR的检测效率。PCR方法在存在其他竞争微生物的情况下表现出特异性，如常规培养方法所证实。没有假阳性或阴性观察时，家庭和环境水样进行了测试invA-PCR分析平行的文化方法。
A polymerase chain reaction (PCR)-based method for the detection of Salmonella species in water samples was optimised and evaluated for speed, specificity and sensitivity. Optimisation of Mg2+ and primer concentrations and cycling parameters increased the sensitivity and limit of detection of PCR to 2.6 x 104 cfu/m.. A 6h non-selective pre-enrichment step further increased the limit of detection to 26 cfu/m.. Out of 14 different Salmonella strains tested, only two, Salmonella arizonae and Salmonella pullorum, did not give positive amplification results with primers homologous to a conserved region of the invA gene. When environmental and drinking waters were assessed, a non-selective pre-enrichment step was included to increase the detection efficiency of PCR. The PCR method demonstrated specificity in the presence of other competing micro-organisms as confirmed by the conventional culture method. No false positives or negatives were observed when household and environmental water samples were tested by invA-PCR analysis parallel to the culture method.