摘要
针对蜡样芽孢杆菌(B.cereus)的hblA基因、产色葡萄球菌(S.chromogenes)的sodA基因和肺炎克雷伯氏菌(K.pneumoniae)的khe基因,建立了三重PCR检测方法。结果显示,退火温度为54℃,BC-hblA、SC-sodA和KP-khe引物浓度分别为0.6、0.2、0.1μmol/L为最佳扩增条件;对B.cereus、S.chromogenes和K.pneumoniae的基因组DNA最低检测限分别为35.8、6.3、40.2 pg/μL,菌液最低检测限分别为9.9×10^(3)、1.1×10^(3)、1.0×10^(4)CFU/mL。特异性试验显示,除这3种菌以外,大肠埃希氏菌、沙门氏菌、金黄色葡萄球菌、链球菌、产气荚膜梭菌和腐败梭菌检测结果均为阴性。14份临床乳房炎乳样的检测结果显示,三重PCR方法B.cereus、S.chromogenes和K.pneumoniae的阳性率分别为35.7%(5/14)、14.3%(2/14)和7.1%(1/14),其中蜡样芽孢杆菌的PCR检测阳性率高于细菌分离鉴定阳性率(28.6%),细菌分离鉴定与PCR检测方法的阳性符合率为87.5%。结果表明,成功建立了能够同时检测蜡样芽孢杆菌、产色葡萄球菌和肺炎克雷伯氏菌的三重PCR方法,为引起乳房炎的病原菌的检测补充了新的方法,并为畜牧生产中这3种细菌的快速检测提供了有效方法。
A triplex PCR assay was developed for detecting the hemolytic gene(hblA)of Bacillus cereus,the superoxide dismutase gene(sodA)of Staphyococcus chromogenes and the hemolytic enzyme gene(khe)of Klebsiella pneumoniae.The results showed that the optimized annealing temperature was 54℃with the optimized concentrations of BC-hblA,SC-sodA,KP-khe primer pairs 0.6μmol/L,0.2μmol/L and 0.1μmol/L,respectively.The thresholds of detection of the triplex PCR assay were 35.8 pg/μL,6.3 pg/μL and 40.2 pg/μL of genomic DNA of B.cereus,S.chromogenes and K.pneumoniae,as well as 9.9×10^(3)CFU/mL,1.1×10^(3)CFU/mL,1.0×10^(4)CFU/mL of bacterial cultures,respectively.The specificity test showed that E.coli,Salmonella,S.aureus,S.treptococcus,C.perfringens,C.septicum were all negative except the 3 pathogens.To detect 14 milk samples from mastitis,the positive rates detected by the triplex PCR of B.cereus,S.chromogenes,K.pneumoniae were 35.7%(5/14),14.3%(2/14),7.1%(1/14),respectively,whereas the positive rate of B.cereus was higher than that of isolation rate(28.6%),the positive coincidence rate between bacterial isolation and triplex PCR detection was 87.5%.In summary,a triplex PCR method was established for the detection of B.cereus,S.chromogenes and K.pneumoniae that may cause bovine mastitis in this study,which provided an alternative method for the quick detection in animal husbandary.
作者
胡慧
李田美
姜艳芬
HU Hui;LI Tian-mei;JIANG Yan-fen(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China)
出处
《动物医学进展》
北大核心
2022年第7期1-6,共6页
Progress In Veterinary Medicine
基金
陕西省重点研发计划项目(2021NY-026)。
