MS2噬菌体在贝类食源性病毒检测中过程质控应用

Application of MS2 phage in process control in detection of food borne virus in shellfishes

目的研究MS2噬菌体作为过程质控品在贝类食源性病毒检测中的应用。方法采用双层琼脂培养法制备MS2噬菌体悬液,计算效价;实时荧光定量逆转录聚合酶链式反应(qRT-PCR)检验其稳定性;比较3种RNA提取方法的提取效率,确定M S2噬菌体的最适添加量范围;分析M S2噬菌体添加对贝类食源性病毒检测的影响。结果稳定性研究表明-20℃和-80℃28 d MS2噬菌体(悬液的效价浓度为7.13×1011pfu/m L)时RNA含量无明显变化[t=0.464,P=0.653(-20℃);t=0.602,P=0.561(-80℃)]。MS2噬菌体最适添加量范围为7.13×108~7.13×107pfu/m L。在最适添加范围内,试剂盒方法和Trizol裂解结合磁珠纯化方法回收率均>1%。2种阳性样品均被检出,其Ct值比较接近,M S2噬菌体对贝类食源性病毒检测无影响(t=0.170,P=0.873)。结论M S2噬菌体可作为贝类食源性病毒qRT-PCR检测的过程质控品,是监控假阴性结果的一种简便有效的方法。

Objective To develop a procedure of MS2 phage application in process control in real-time quantitative reverse transcription-PCR （qRT-PCR） detection of food borne virus in shellfishes. Methods MS2 phage suspension was prepared using double-agar-layer plaque technique. The stability of the phages was tested by applying qRT-PCR. Using the methods referred by International Organization for Standardization （ISO）, MS2 phages were added to shellfish matri- ces,and the RNA was extracted by three methods. RNA was detected with qRT-PCR and a standard curve was estab- lished. The adding amount of MS2 phage was optimized after calculating and comparing the recovery rate of three RNA extract methods. Based on the test results of two artificially constructed positive samples, the impact of MS2 phage on food borne virus detection was analyzed. Results When the titer of MS2 phage suspension was 7. 13 x 10~ plaque form- ing unit （pfu）/ml, no significant variation in RNA content of MS2 phage was observed in the suspension kept at the tem- perature of - 20 ^（2 （ t = 0. 464 ,P = O. 653 ） or - 80 ~C （ t = 0. 602 ,P = 0. 561 ） for 28 days. The optimal additive amount of MS2 phage ranged between 7.13 x 108 and 7. 13 x 107 pfu. The RNA recovery rate was greater than 1% when RNA was extracted with commercial kit or method of trizol combined with magnetic beads purification, which meet the require- ments of using MS2 phage as a process control. Two artificial samples were both detected with positive results and the cy- cle threshold （Ct） values of the detections were very close to each other （t =0. 170,P =0. 873）. Thus,MS2 phage had no influence on detection of virus in shellfishes. Conclusion Employing MS2 phage as a process control in qRT-PCR detection of food-borne virus in shellfish could be a simple and effective way to monitor false negative results.