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比较 2 种培养方法和 PCR 检测法在禽类粪便中沙门氏菌检测的应用。

Comparison of 2 culture methods and PCR assays for Salmonella detection in poultry feces.

机构信息

Instituto Nacional de Tecnología Agropecuaria (INTA), Estación Experimental Agropecuaria, Concepción del Uruguay, Casilla de Correo Nº6, 3260, Entre Ríos, Argentina.

出版信息

Poult Sci. 2012 Mar;91(3):616-26. doi: 10.3382/ps.2011-01831.

Abstract

The present work compared 2 culture methods and the combinations of pre-enrichment and enrichment culture methods with PCR assays [buffered peptone water-PCR and tetrathionate-PCR or modified semisolid Rappaport-Vassiliadis (MSRV)-PCR] for motile and nonmotile Salmonella strain detection using artificially contaminated poultry feces. The specificity and positive predictive values were equal to one in both culture methods. Specificity and positive predictive values, accuracy, sensitivity, and negative predictive values were higher for motile than nonmotile Salmonella strains in culture methods. Only Salmonella enterica serovar Gallinarum was detected by the MSRV method with low accuracy, sensitivity, and negative predictive value. The detection level of motile strains was 2 ×10(0) to 22 × 10(2) cfu per 25 g for these methods, whereas it was 6.9 × 10(2) cfu per 25 g in culture methods for Salmonella Gallinarum. Extending the incubation time of the enrichment medium to 6 d in the TT method did not improve the isolation rates. In general, all selective plating media did not show any statistical differences in the parameters of performance studied. On the other hand, accuracy and sensitivity values were higher in MSRV-PCR and tetrathionate-PCR methods than in the buffered peptone water-PCR method. Specificity and positive predictive values were equal to one in most of the cases. In terms of detection limits, motile Salmonella strains were recovered from 5 × 10(0) cfu per 25 g in MSRV-PCR and tetrathionate-PCR methods, whereas the detection limit was better for nonmotile Salmonella in MSRV-PCR methods than in the tetrathionate-PCR method. Kappa coefficients showed that there was a very good agreement between tetrathionate and MSRV methods for motile Salmonella strains, whereas these methods did not show any concordance for nonmotile Salmonella strains. When buffered peptone water-PCR was compared with both tetrathionate-PCR and MSRV-PCR, agreement was poor for motile Salmonella strains and slight to fair for nonmotile Salmonella strains. The difference in isolation rate obtained with the methods used for motile and nonmotile Salmonella strains must be taken into account when a poultry fecal sample is considered negative for the presence of Salmonella.

摘要

本研究比较了 2 种培养方法以及预富集和富集培养方法与聚合酶链反应(缓冲蛋白胨水-PCR、亚硒酸盐-四硫酸盐增菌液-PCR 或改良半固体 Rappaport-Vassiliadis 增菌液-PCR)联合检测人工污染禽粪中运动性和非运动性沙门氏菌的效果。两种培养方法的特异性和阳性预测值均为 1。在培养方法中,运动性沙门氏菌的特异性、阳性预测值、准确性、敏感性和阴性预测值均高于非运动性沙门氏菌。MSRV 方法对肠炎沙门氏菌的检测特异性、敏感性和阴性预测值均较低。运动性菌株的检测水平为这些方法每 25 g 粪便中 2×10(0)至 22×10(2) cfu,而沙门氏菌肠炎每 25 g 粪便中为 6.9×10(2) cfu。在 TT 法中延长增菌时间至 6 d 并没有提高分离率。一般来说,所有选择性平板培养基在研究的性能参数方面没有显示出任何统计学差异。另一方面,MSRV-PCR 和亚硒酸盐-四硫酸盐增菌液-PCR 方法的准确性和敏感性值均高于缓冲蛋白胨水-PCR 方法。在大多数情况下,特异性和阳性预测值均为 1。在检测限方面,运动性沙门氏菌在 MSRV-PCR 和亚硒酸盐-四硫酸盐增菌液-PCR 方法中可从 5×10(0) cfu/25 g 粪便中回收,而 MSRV-PCR 方法对非运动性沙门氏菌的检测限优于亚硒酸盐-四硫酸盐增菌液-PCR 方法。kappa 系数表明,运动性沙门氏菌在亚硒酸盐和 MSRV 方法之间具有非常好的一致性,而对于非运动性沙门氏菌,这两种方法之间没有一致性。当缓冲蛋白胨水-PCR 与亚硒酸盐-四硫酸盐增菌液-PCR 和 MSRV-PCR 进行比较时,运动性沙门氏菌的一致性较差,而非运动性沙门氏菌的一致性为轻度到中度。在考虑禽粪样是否为沙门氏菌阴性时,必须考虑用于运动性和非运动性沙门氏菌的方法之间的分离率差异。

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