Effects of Primers and Taq Polymerase on Randomly Amplified Polymorphic DNA Analysis for Typing Listeria monocytogenes From the Environment of a Shrimp Processing Plant

Ninety-nine randomly selected isolates of Listeria monocytogenes from several processing environment locations, in a shrimp processing plant, obtained during a 5-month sampling period were subjected to randomly amplified polymorphic DNA (RAPD) analysis with the use of four primers. Preliminary studies indicated that the number of DNA bands and their intensity differed greatly with respect to the commercial source of the Taq polymerase used with individual isolates. Eighteen composite RAPD types were discerned with the use of the four primers. Among these 18 composite RAPD types, type 1 comprised 14 indistinguishable isolates, and type 9 comprised 49 indistinguishable isolates. These results indicate that the shrimp processing plant was dominated by these 2 RAPD types that comprised 63.6% of the 99 randomly selected isolates.     

单核细胞增生性李斯特菌毒力基因检测及DNA随机扩增多态性分型

为了解在昌平地区分离的单核细胞增生性李斯特菌（Lm）携带毒力基因的状况和DNA随机扩增多态性（RAPD）分型情况，采用聚合酶链反应（PCR）对28株加进行了溶血素基因（hfy）、与侵袭性有关的卸和prfA基因的测定及RAPD分型。结果3个毒力基因PCR全部呈阳性反应，两株无害加为阴性反应。用RAPD分型，28株加分为12型．A、B两型共9株菌全部来自某肉联厂；C、D两型共10株来自某肉鸡公司；另外9株加有8个RAPD型．分别来自熟肉制品及零售市场。此次实验发现昌平地区在不同时间、不同地点分离的加其RAPD型别变化较大。有多种型别的细菌存在。根据RAPD分型看出肉联厂与肉鸡公司可能存在着内部污染问题。     

Application of Random Amplified Polymorphic DNA (RAPD) and Pulsed-Field Gel Electrophoresis (PFGE) Analysis to Listeria monocytogenes: A Review of Methodology and Results

Random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) analyses have been found to be powerful molecular methods for differentiating isolates of a given bacterial species. When applied to Listeria monocytogenes, both methods have been found highly effective in tracking isolates involved in food borne outbreaks of listeriosis and in identifying routes of contamination in food processing plants. Among the two methods, PFGE is considered somewhat superior in discriminatory power. However, the use of two or more independent random primers with RAPD is considered to result in a level of discrimination equal to that of PFGE. When results from both methods are combined, a maximum level of discrimination that exceeds that obtained with both methods independently can be achieved. Individually, both methods far exceed the discriminatory power of serotyping and phage typing of L. monocytogenes strains in that serotypes 1/2a, 1/2b, and 4b, represent over 90% of all human isolates, and phage typing at times has allowed typing of no more than about 50% of isolates. In addition, both RAPD and PFGE on occasion have been found to be superior to ribotyping, multilocus enzyme electrophoresis, and restriction enzyme analysis of L. monocytogenes isolates.     

低温条件下即食虾中单增李斯特菌与副溶血性弧菌共存分子预测模型的建立

本研究旨在应用实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction,qPCR）技术描述即食虾中的单增李斯特菌与副溶血性弧菌的生长行为,构建混菌模式下的分子预测模型。将单增李斯特菌与副溶血性弧菌等量（（5.0±0.5）（lg（CFU/g）））混合接种于即食虾中,置于低温环境（4 ℃和10 ℃）下培养,并利用qPCR定量检测单增李斯特菌与副溶血性弧菌数量的动态变化。运用生长模型（修正Gompertz、Logistic、Baranyi）和失活模型（Log-linear、Weibull）分别拟合两株菌的生长和失活趋势。结果表明：低温条件下,修正Gompertz、Logistic和Baranyi模型均可成功拟合单增李斯特菌的生长曲线,其决定系数（R2）均大于0.98。对于副溶血性弧菌,在4 ℃条件下,Log-linear和Weibull模型能够清晰地描述其失活情况,R2分别为0.950和0.945;而10 ℃条件下,2 个失活模型均难以描述其行为变化,R2仅为0.784和0.775。本研究运用分子生物学技术描述即食虾中两种致病菌混合培养的菌量变化,探究混菌模式下微生物的生长失活情况,为分子预测模型的进一步研究提供了新的思路。