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

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 matrices,and the RNA was extracted by three methods.RNA was detected with qRT-PCR and a standard curve was established.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 7.13×10¹¹ plaque forming unit (pfu)/ml,no significant variation in RNA content of MS2 phage was observed in the suspension kept at the temperature of-20℃ (t=0.464,P=0.653) or-80℃ (t=0.602,P=0.561) for 28 days.The optimal additive amount of MS2 phage ranged between 7.13×10⁸ and 7.13×10⁷ 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 requirements of using MS2 phage as a process control.Two artificial samples were both detected with positive results and the cycle 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.