NASBA (Nucleic Acid Sequence Based Amplification) detection primer, probe and detection method for GII type norovirus in sea foods
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A technology for virus detection and food, applied in the technical field of rapid detection of GII type norovirus in marine food, achieving high specificity and sensitivity, simple operation steps, and short detection time
Pending Publication Date: 2014-01-22
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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Although the symptoms of acute gastroenteritis (vomiting, diarrhea, and abdominal cramps) caused by norovirus are self-limiting, it can still lead to death if not treated in time. The norovirus method is conducive to improving the quality and safety of marine food in my country, and is also conducive to protecting the health of consumers
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Embodiment 1
[0035] A GII type norovirus detection primer,
[0036] The forward primer Nor-F sequence is SEQ ID NO1: 5'-TGGAATTCCATCGCCCACT-3';
[0037] The reverse primer Nor-R sequence is SEQ ID NO2:
[0042] The molecular beacon probe is labeled with a fluorescent reporter group FAM at the 5' end, and a fluorescent quencher group DABCYL at the 3' end.
Embodiment 3
[0044] A method for quantitative detection of GII type norovirus, comprising the following steps:
[0045] (1) Construction of GII norovirus standard cRNA
[0046] Using the above primers, the GII norovirus was amplified and optimized by conventional RT-PCR, and the specific product fragment size was 119bp; then the target fragments were cloned, plasmid extracted, linearized by enzyme digestion, in vitro transcription and cRNA accurate Sexuality verification, determination of CopyRNA concentration and copy number calculation; after 10-fold serial dilution of GII norovirus RAN, aliquot it as a standard and store it at -80°C;
[0047] (2) Real-time NASBA response
[0048] (1) To the standard substance preserved in step (1), make 9 gradient concentration standard substance cRNAs altogether, are respectively 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 copy; and extract RNA in the sample to be tested, wherein the concentration of RNA is 50-100ng / μL;
[0...
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Abstract
The invention discloses an NASBA (Nucleic Acid Sequence Based Amplification) detection primer, a probe and a detection method for GII type norovirus in sea foods. According to the NASBA detection primer, a forward primer sequence is represented as SEQ ID NO.1 and a backward primer sequence is represented as SEQ ID NO.2; the primer sequence of a molecular beacon probe for detecting the GII type norovirus is represented as SEQ ID NO.3. The detection method comprises the following steps: (1) constructing a standard cRNA (complementary Ribonucleic Acid) of the GII type norovirus; (2) carrying out a real-time NASBA reaction; (3) qualitatively and quantitatively analyzing. The primer and the probe disclosed by the invention have high specificity on the detection of the GII type norovirus. The detection method is simple in operation steps and short in detection time, can be used for carrying out a qualitative and quantitative analysis and has high specificity and sensitivity.
Description
technical field [0001] The invention relates to a technology for rapid detection of GII type norovirus in marine food based on real-time nucleic acid sequence-based amplification (Nucleic acid sequence-based amplification, NASBA) technology. Background technique [0002] Norovirus (NVs) is a virus that exists in marine food and can cause acute gastroenteritis in adults and children. The norovirus genome is a single-stranded positive-sense RNA with a full length of 7642 nt, including three open reading frames (open reading frames, ORFs) ORFs1, ORFs2 and ORFs3. ORFs1 encodes a conserved non-structural protein with RNA polymerase activity; ORFs2 encodes a capsid protein with a molecular weight of about 56Kda; ORFs3 encodes a strongly basic microstructural protein with a molecular weight of about 22.5Kda, and some regions interact with the capsid protein , together forming the capsid. People are infected with norovirus mainly through contact with contaminated food and water so...
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