唐菖蒲伯克霍尔德氏菌椰毒致病型实时荧光PCR方法的建立

Developing a Novel Method Detecting Burkholderia Gladioli Pv. Cocovenenans by Real-Time Fluorescent PCR

目的:建立唐菖蒲伯克霍尔德氏菌椰毒致病型菌株(Burkholderia gladioli pv.cocovenenans)的实时荧光PCR方法。方法:根据唐菖蒲伯克霍尔德菌椰毒致病型菌株Co14的米酵菌酸生物合成基因bonM序列,用Primer Premier 6设计一对特异性引物和一个TaqMan探针,建立了实时荧光PCR反应体系。通过对5株唐菖蒲伯克霍尔德氏菌椰毒致病型菌株及14株其他菌株进行实时荧光PCR检测,验证该方法的特异性。同时从菌液浓度和DNA浓度水平上进行研究,确定该检测方法的灵敏度。并同时采用该方法和GB 4789.29—2020,对155粉湿米粉及其原料大米进行检测,验证该方法的实用性。结果:实时荧光PCR方法特异性检测结果显示,5株唐菖蒲伯克霍尔德氏菌椰毒致病型菌株均扩增阳性,其他菌株均扩增阴性。通过灵敏度试验,确定该方法检测唐菖蒲伯克霍尔德氏菌椰毒致病型菌株的菌液检测灵敏度为6.5×10^(2)cfu/mL,DNA检测灵敏度为4.8×10^(-4)ng/μL。实际样品的检测结果显示,采用该方法进行检测,2份湿米粉和3份大米PCR检测阳性,其余150份样品均为阴性,这与采用GB 4789.29—2020检测的结果一致,验证了该检测体系的实用性。结论:文章建立的实时荧光PCR检测方法,反应时间仅需1 h,设计的引物和探针具有较高的特异性及灵敏度,能够有效地用于唐菖蒲伯克霍尔德氏菌椰毒致病型菌株的快速筛查,这为唐菖蒲伯克霍尔德氏菌椰毒致病型菌株的检测提供了一种简便、快速、准确的分子检测手段。

Objective:A real-time fluorescent PCR method was to be established for detection of Burkholderia gladioli pv.cocovenenans.Methods:According to the bonM sequences of the bongkrekic biosynthesis gene of Burkholderia gladioli pv.cocovenenans strain Co14,a pair of specific primers and a TaqMan probe were designed with Primer Premier 6,and a real-time fluorescent PCR reaction system was established.To verify the specificity of this method,5 strains of Burkholderia gladioli pv.cocovenenans and 14 other strains were detected by real-time fluorescent PCR.At the same time,the sensitivity of the detection method was determined by studying the concentration of the bacterial solution and the DNA concentration.And in order to verify the practicability of this method,155 wet rice noodles and its raw rice samples were detected using the established methods compared with method provided from national standard GB 4789.29—2020.Result:The specific detection results of real-time fluorescent PCR method showed that the 5 Burkholderia gladioli pv.cocovenenans strains were all amplified positively,whereas the other strains were all negatively amplified.The sensitivity test results of real-time fluorescent PCR method showed that the detection limits of DNA and bacterial suspension were 4.8×10^(2) ng/μL and 6.5×10^(-4) cfu/mL,respectively.Using real-time fluorescent PCR method,2 wet rice noodles and 3 rice PCR tests were positive,and the remaining 150 samples were all negative which were 100% consistent with the results of the GB 4789.29—2020.The test results of the actual samples verified the practicability of detection system.Conclusion:In this study,the total reaction time of real-time fluorescent PCR was only about 1 hour,and the primers and probe designed had high specificity and sensitivity which could be used for rapid screening of Burkholderia gladioli pv.cocovenenans effectively.This provided a simple,fast and accurate molecular detection method for Burkholderia gladioli pv.cocovenenans.