Burkhalter P W, Müller C, Lüthy J, Candrian U
University of Bern, Institute of Biochemistry, Switzerland.
J AOAC Int. 1995 Nov-Dec;78(6):1531-7.
A polymerase chain reaction (PCR) method for direct detection of Salmonella spp. in whole-shell eggs is described. The method does not require isolating strains. Preenrichment, rapid DNA isolation, and a nested PCR system targeting the invA gene enabled detection of the genus Salmonella. The specific nested PCR product of 283 base pairs was formed from all 21 serovars, including 43 Salmonella strains tested. No PCR product was formed from 56 non-Salmonella enterobacteriacea and other bacterial strains tested. Experiments with artificially contaminated eggs showed a detection limit of about 10(3)-10(4) colony-forming units (cfu)/egg before and about 1-10 cfu/egg after preenrichment. In analysis of 180 single eggs from 4 flocks and 36 pools of 5 eggs each from another 4 flocks from the same producer, Salmonella spp. were detected in 5 of 90 eggs from 2 different flocks. Determination of anti-Salmonella antibodies in eggs yielded positive results for 2 additional and the 2 PCR-positive flocks. In contrast, classical selective culture detected Salmonella spp. in only 1 of 100 eggs in one flock when 100 eggs from each flock were analyzed.
本文描述了一种用于直接检测全蛋液中沙门氏菌属的聚合酶链反应（PCR）方法。该方法无需分离菌株。预富集、快速DNA分离以及针对invA基因的巢式PCR系统能够检测沙门氏菌属。从包括测试的43株沙门氏菌在内的所有21个血清型中均形成了283个碱基对的特异性巢式PCR产物。测试的56株非沙门氏菌肠杆菌科细菌和其他细菌菌株均未形成PCR产物。对人工污染鸡蛋的实验表明，预富集前的检测限约为10(3)-10(4)菌落形成单位（cfu）/蛋，预富集后的检测限约为1-10 cfu/蛋。在对来自4个鸡群的180个单个鸡蛋以及来自同一生产商的另外4个鸡群的36组每组5个鸡蛋进行分析时，在来自2个不同鸡群的90个鸡蛋中的5个中检测到了沙门氏菌属。对鸡蛋中抗沙门氏菌抗体的测定在另外2个鸡群以及2个PCR阳性鸡群中产生了阳性结果。相比之下，当对每个鸡群的100个鸡蛋进行分析时，经典的选择性培养仅在一个鸡群的100个鸡蛋中的1个中检测到了沙门氏菌属。
