11种致牛腹泻病原的一步法多重PCR/RT-PCR检测方法的建立

A one-step multiplex PCR/RT-PCR method for detecting 11 pathogens causing bovine diarrhea

摘要:

【背景】单病原或多病原混合感染导致的牛腹泻综合征严重制约养牛业发展，病原快速准确诊断是防控该病的重要前提。【目的】建立11种致牛腹泻的细菌或病毒的一步法多重PCR/RT-PCR检测方法，实现快速诊断病原。【方法】通过文献检索调查了引起牛腹泻病的主要病原及其高覆盖率的引物，选择以产气荚膜梭菌(Clostridium perfringens) α-toxin、肠道沙门氏菌(Salmonella enterica) invA、大肠杆菌(Escherichia coli) K99、牛肠道病毒(bovine enterovirus, BEV) 5'-UTR、牛星状病毒(bovine astrovirus, BAstV)ORF1a、牛轮状病毒(bovine rotavirus, BRoV)VP6、牛嵴病毒(bovine kobuvirus, BKoV) D4、牛诺如病毒(bovine norovirus, BNoV) ORF1、牛冠状病毒(bovine coronavirus, BCoV) N、牛环曲病毒(bovine torovirus, BToV) N、牛病毒性腹泻黏膜病病毒(bovine viral diarrhea virus, BVDV) 5'-UTR为靶标序列的引物，通过温度梯度PCR及单一控制变量法优化退火温度、引物浓度、循环数，建立11种病原的多重PCR/RT-PCR检测方法，对该方法进行特异性、灵敏度和重复性评价，并应用该方法进行临床样品检测。【结果】最佳退火温度为54.4 ℃，C. perfringens、S. enterica、E. coli、BEV、BAstV、BRoV、BKoV、BNoV、BCoV、BToV和BVDV对应基因片段最优引物浓度分别为0.20、0.25、0.25、0.20、0.25、0.25、0.35、0.50、0.25、0.25、0.30 μmol/L，最优循环数为35个循环。该方法特异性强，仅对靶标病原检测为阳性，对溶血性曼氏杆菌(Mansiella haemolyticus)、化脓链球菌(Streptococcus pyogenes)、牛支原体(Mycoplasma bovine)分离株等病原检测均为阴性；敏感性高，对重组质粒标准品最低检出限依次为7.5×10³、7.5×10⁴、7.5×10³、7.5×10¹、7.5×10⁴、7.5×10²、7.5×10³、7.5×10²、7.5×10²、7.5×10⁴、7.5×10³ copies/μL；重复性好，批间与批内试验均一致。用该方法检测江苏地区临床样品490份，结果显示，BEV、BAstV、BRoV、BKoV、BNoV、BCoV、BToV、BVDV阳性率分别0.61%、0.41%、0.61%、0.21%、27.14%、3.27%、0.21%、1.02%。通过单重PCR对该多重PCR临床样品检测结果进行重复验证，结果显示符合率为100%。随机挑选50个检测为阳性的PCR产物进行测序验证，结果均为相应病原的基因片段。【结论】建立一种同时检测11种牛腹泻主要病原的一步法多重PCR/RT-PCR检测方法。

Abstract:

[Background] The bovine diarrheal syndrome, caused by single or multiple pathogen infections, significantly restricts the development of the cattle industry. Rapid and accurate pathogen diagnosis is crucial for disease control. [Objective] To establish a one-step multiplex PCR/RT-PCR method for detecting 11 bacterial and viral pathogens causing bovine diarrhea, facilitating rapid pathogen diagnosis. [Methods] We identified the main pathogens causing bovine diarrhea and appropriate high-coverage primers by literature searching. According to the searching results, we selected primers for the following target genes: Clostridium perfringens α-toxin, Salmonella enterica invA, Escherichia coli K99, bovine enterovirus (BEV) 5'-UTR, bovine astrovirus (BAstV) ORF1a, bovine rotavirus (BRoV) VP6, bovine kobuvirus (BKoV) D4, bovine norovirus (BNoV) ORF1, bovine coronavirus (BCoV) N, bovine torovirus (BToV) N, and bovine viral diarrhea virus (BVDV) 5'-UTR. After optimization of the annealing temperature with the temperature gradient PCR method as well as the primer concentration and cycle number with the single factor test method, we established a one-step multiplex PCR/RT-PCR method for detecting the 11 pathogens. The specificity, sensitivity, and repeatability of the method were evaluated, and then the method was employed to detect clinical samples. [Results] The optimal annealing temperature was 54.4 ℃. The optimal concentrations of primers for the target genes in C. perfringens, S. enterica, E. coli, BEV, BAstV, BRoV, BKoV, BNoV, BCoV, BToV, and BVDV were 0.20, 0.25, 0.25, 0.20, 0.25, 0.25, 0.35, 0.50, 0.25, 0.25, and 0.30 μmol/L, respectively. The optimal number of cycles was 35. The established method demonstrated high specificity, yielding positive results only for the target pathogens and negative results for Mannheimia haemolytica, Streptococcus pyogenes, and Mycoplasma bovine. This method exhibited high sensitivity, with the lowest limits of detection of the recombinant plasmid standards being 7.5×10³, 7.5×10⁴, 7.5×10³, 7.5×10¹, 7.5×10⁴, 7.5×10², 7.5×10³, 7.5×10², 7.5×10², 7.5×10⁴, and 7.5×10³ copies/μL, respectively. The method showed good reproducibility, with consistent inter-batch and intra-batch test results. We then used the established method to detect 490 clinical samples from Jiangsu. The positive rates for BEV, BAstV, BRoV, BKoV, BNoV, BCoV, BToV and BVDV were 0.61%, 0.41%, 0.61%, 0.21%, 27.14%, 3.27%, 0.21%, and 1.02%, respectively. The results of the one-step multiplex PCR/RT-PCR method in clinical samples were consistent with those of single PCR. Sequencing verification of 50 randomly selected positive PCR products confirmed the presence of genes corresponding to the identified pathogens. [Conclusion] We successfully established a one-step multiplex PCR/RT-PCR method for the simultaneous detection of 11 major pathogens causing bovine diarrhea.