牛羊多杀性巴氏杆菌多重TaqMan qPCR检测方法的建立
Establishment of multiple TaqMan qPCR assay for Pasteurella multocida in cat-tle and sheep
郭亚男 1张正刚 2王建东 1王景松 2李珂 2李继东 3梁小军1
作者信息
- 1. 宁夏农林科学院动物科学研究所,宁夏银川 750002
- 2. 宁夏农林科学院动物科学研究所,宁夏银川 750002;宁夏大学动物科技学院,宁夏银川 750021
- 3. 宁夏大学动物科技学院,宁夏银川 750021
- 折叠
摘要
为建立多杀性巴氏杆菌多重TaqMan荧光定量PCR检测方法,根据NCBI数据库中多杀性巴氏杆菌hyaC-hy-aD、bcbD、dcbF、ecbJ、fcbD 5种荚膜基因序列设计特异性引物和荧光标记探针,通过梯度设置调整退火温度,用矩阵法对引物与探针浓度进行优化,构建标准曲线,进行特异性、灵敏度及重复性试验,最终建立针对这5种基因的多重TaqMan qPCR检测方法.结果显示,建立的检测方法其扩增曲线具有良好的线性关系;灵敏度较高,比普通PCR高10～100倍;特异性强,对芽孢杆菌、奇异变形杆菌、金黄色葡萄球菌、放射根瘤菌等8种病原菌DNA检测均无扩增曲线;组间及组内重复性试验Ct值变异系数均小于3％;通过对90份临床样本进行检测,显示该检测方法比常规PCR检测方法检出率高出11.25％.结果表明本研究建立的方法能够快速、高效地检测多杀性巴氏杆菌及其荚膜分型,对临床和实验室的快速诊断具有重要意义.
Abstract
This study aims to establish a multiplex TaqMan fluorescence quantitative PCR(qPCR)assay for Pasteurella multocida(P.multocida).Specific primers and fluorescent labeling probes were designed based on the sequences of five podoplanar genes of P.multocida hyaC-hyaD,bcbD,dcbF,ecbJ,and fcbD in the NCBI database.We adjusted the annealing temperature by gradient setting,optimized the primer and probe concentrations by matrix method,constructed standard curves,and performed specificity,sensitivity and reproducibility tests,and finally established mul-tiplexed TaqMan qPCR assays for these five genes.The results showed that the established assay had a good linear relationship between the amplification curves.The sensitivity of this method was high,10-100 times higher than that of ordinary PCR;the specificity was strong,and there was no amplification curve in the DNA detection of eight pathogenic bacteria such as Bacillus,Proteus mirabilis,Staphylococcus aureus,and Rhizoctonia rad iodurans.This assay had a good linear rela-tionship,and the coefficients of variation for Ct values of the inter-and intra-group reproducibility tests were all less than 3％,and the detection rate of this assay was 11.25％higher than the con-ventional PCR assay through the detection of 90 clinical samples.The method established in this study is able to detect P.multocida rapidly and sensitively,which is important for its rapid clinical and laboratory diagnosis.
关键词
牛羊/多杀性巴氏杆菌/荚膜分型/多重TaqMan荧光定量PCR/检测方法Key words
cattle and sheep/Pasteurella multocida/capsule typing/multiple TaqMan qPCR/detec-tion method引用本文复制引用
出版年
2024
NETL
NSTL
万方数据