Study On Reverse Transcription-Polymerase Chain Reaction Assay For Detection Of Norwalk-like Virus In Shellfish

Norwalk-like virus has been considered one of the most important reasons of nonbacterium acute gastroenteritis. In the word, there are many cases that are caused by NLVs. Foods that are frequently incriminated in NLVs food poisoning include shellfishes vegetables ,salad and so on. In this study , a rapid method has been established to detect NLVs in food, increase the sensitivity of method and provide a technical help.In this study, MgCl₂ concentration, annealing temperature ,primer concentration and PCR circles are optimized to determine the optimal PCR. The reaction mixture consisted of RT mixture and PCR mixture. RT mixture consisted of 1μL of AMV(5U ? μL^-1) , 4μL of 5×Reverse transcriptase buffer, 0.5μL of RNase Inhibitor (40U·μL^-1), 2μL of mixture of dNTPs(the 10 mmol ? L^-1 each), 3.5μL of downstream primer (10μmol ? L^-1),4μL of RNA,5μL of double-distilled DEPC H₂O;PCR mixture consisted of 20μL of RT-Mixture,5μL of 10 × PCR buffers, 1μL of Taq(5U·μL^-1), 5uL of mixture of dNTPs( the 2.5 mmol ? L^-1 each), 2μL of upstream primer(10nmol ? L^-1), 3.5μL of Mg²⁺(25 mmol ? L^-1),10μL of double-distilled water. The reaction was run under the following conditions: Reverse transcription at 42 ℃ for lh;DNA pre-denaturation at 94℃ for 3min;DNA denaturation at 94℃ for 0.5 min ,primer annealing at 46.9℃ for 1.5 min ,and DNA extension at 72℃ for 1.0 min for 40 cycles;the last extension was performed at 72℃ for 5 min.The PCR products were examined by electrophoresis with 2% agarose gel. A DNA fragment of 327 bp was amplified. PCR products were confirmed by DNA sequencing. The sensitivity of detection was 79 RT-PCR₅₀.The effect of 4 methods of concentrating virus from shellfish were compared. A efficient concentration method was confirmed for extraction of NLVs from shellfish. That method precipitated virus to remove RT-PCR inhibitors The effect of 4 methods of extracting RNA from NLVs in shellfish were compared. A efficient extraction procedure was confirmed for extraction of NLVs RNA from shellfish. That method can extract the more pure RNA, improved the sensitivity. The lower detection limit was 8.1 × 10² RT-PCR₅₀/5g shellfish. The whole procedure needed 11 hours.In this study, 3 of 25 samples were detected that were contaminated with NLVs. The product of RT-PCR of the positive sample were confirmed by DNA sequencing.The resultsof Blastn of 3 isolates in this study indicate are with high homology percent to the strain DQ369797.1, 92%⁹4%.The positive sample that explain to examine is the Norwalk-like virus, verifying originally experiment result of method.