Development Of Standard Sample Of Tow Food-bore Viruses In Shellfish

Food-borne virus is a kind of virus caused by food and generally can lead to virus diseases.Oysters and other shellfish products have strong enrichment because of its filter feeding characteristics.However,there are still a lot of problems in the detection and quantification of the virus,which usually use the clinic diarrhea samples and inactivated vaccine,by the lack of uniform standard samples.In this article,based on the armored RNA technology and using Qbeta developing positive standard samples of Norovirus and hepatitis A virus,there are important practical significances on improving the credibility and accuracy of the detection,ensuring our nation's food safety,promoting the international trade of China's aquatic products,avoiding barriers on international trade.1.Prepare QIEVGII and QGBHAV fragment containing Qbeta maturase coding gene and capsid protein coding genes,packing site,detection target cDNA sequence of GII norovirus and HAV auxiliary polyclonal sites respectively by artificial synthetic way.Then,clone the QINVGII and QGBHAV fragments into pET-28a(+)vector respectively using recombinant enzyme.The recombinant plasmid pET-QINVGII and pET-QGBHAV were successfully constructed through Enzyme digestion reaction and sequencing identification.2.Respectively transfer the recombinant plasmid pET-QINVGII and pET-QGBHAV into BL21 E.coli using the prokaryotic expression system of E.coli,and determine the best expression,which are 0.8mM of final IPTG concentration and 12-hour induction in temperature of 37?.The target protein validated by SDS-PAGE is in 14.4kDa;the diameter of VLPs detected through electron microscope is 27nm,which is consistent with the expected results.At last,confirm that the capsid protein expressed successfully and can be assembled into VLPs.3.Through ultrasonic breaking bacteria,use CsCl density gradient centrifugation and purification after digestion of DNase I and Rnase A.Without Recombinant plasmid residue,the two virus particles prepared by the method can be used for real-time PCR.4.Using real-time PCR,we successfully confirm that the packaging nucleic acid of virus particles are norovirus and hepatitis A virus RNA respectively.Dilute the virus RNA with gradient,and it shows a good linear relationship proved by RT-qPCR.What's more,its detection limit was 10 copies per reaction,with good repeatability.So it can be used for standard sample in virus detection.Constant value results show that the content of prepared virus like particles,containing GII norovirus,is 2×109 copies/?L and coefficient of variation between bottle is 3.48%;The same values of hepat:itis A virus are 3×109 copies/?L and 4.26%.5.Dilute the two kinds of prepared virus-like particles to 2x 107 copies/?L and 106 copies/?L and place them in different conditions for stability study.The stability test results show that:the liquid state sample can be stored stably for half a month at 37 degrees Celsius,25 days at room temperature,5 months at 2-8 degrees,and at least 5 months at-20.The two standard samples of VLPs prepared in this research,respectively containing G? norovirus and HAV detection nucleic acid sequences,can replace the present quality control product prepared from clinical diarrhea samples and inactivated virus vaccine.It's also applicable to detection of virus in disease control,quality inspection,immigration,scientific research and other multiple fields.