Okamura Masashi, Ohba Yousuke, Kikuchi Shuichi, Takehara Kazuaki, Ikedo Masanari, Kojima Tadashi, Nakamura Masayuki
Laboratory of Zoonoses, Kitasato University School of Veterinary Medicine, Towada, Aomori 034-8628, Japan.
Avian Dis. 2009 Jun;53(2):216-21. doi: 10.1637/8450-081808-Reg.1.
The present study developed a loop-mediated isothermal amplification (LAMP) assay that amplifies the fragments of O4 Salmonella enterica-specific gene rfbJ and evaluates the potential use in detection of Salmonella enterica serovar Typhimurium (ST). The detection limit of the LAMP assay was 10(3) CFU/ml, which was lower than that of the PCR assay with the same target gene (10(5) CFU/ml), confirmed by electrophoresis. The increased turbidity of the final products of LAMP was also observed with more than 10(3) CFU/ml. Furthermore, the LAMP assay took only 60 min for a reaction, while the PCR assay needed 80-90 min for a reaction and approximately 30 min for the subsequent electrophoresis to confirm the specific band. The positive reaction was only observed for 55 strains of 11 serovars of O4 group Salmonella enterica. The LAMP assay developed in the present study is considered to be an effective method for specific detection of the O4 group Salmonella enterica serovars, including ST.
本研究开发了一种环介导等温扩增（LAMP）检测方法，该方法可扩增肠炎沙门氏菌O4特异性基因rfbJ的片段，并评估其在检测鼠伤寒沙门氏菌（ST）中的潜在应用。经电泳证实，LAMP检测方法的检测限为10³CFU/ml，低于相同靶基因的PCR检测方法（10⁵CFU/ml）。当菌液浓度超过10³CFU/ml时，还可观察到LAMP最终产物的浊度增加。此外，LAMP检测反应仅需60分钟，而PCR检测反应需要80 - 90分钟，后续电泳确认特异性条带还需约30分钟。仅在11种O4群肠炎沙门氏菌血清型的55株菌株中观察到阳性反应。本研究开发的LAMP检测方法被认为是特异性检测包括ST在内的O4群肠炎沙门氏菌血清型的有效方法。
