Nested PCR primer for detection of GII type norovirus and application thereof
A technology of nested primers and RT-PCR, which is applied in the field of virus detection and biology, can solve the problems of no GII type norovirus primers and methods, and achieve the effects of increasing the positive rate, avoiding false negatives, and avoiding false positives
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Embodiment 1
[0043] Example 1 Primer Sequence and Specificity Evaluation
[0044] According to the GII norovirus typing sequence, nested PCR universal primers for oyster samples were designed. The designed primers include two sets of primer pairs, namely RT-PCR amplification primer pair (one-step reverse transcription) and nested PCR amplification primer pair.
[0045] The upstream (NOG2F) sequence of the nested PCR amplification primer pair: ggagggcgatcgcaatct; the downstream (NOG2R) sequence: ccngcatrvccrttrtacat. As shown in SEQ ID No.1 and 2.
[0046] The upstream (COG2F) sequence of the RT-PCR amplification primer pair: cargarbcnatgttyagrtggatgag; the downstream (NOG2R) sequence: ccngcatrvccrttrtacat. As shown in SEQ ID No.3 and 2.
[0047] Among them, r, b, n, y, h and v are degenerate bases: r represents a or g; b represents c or g or t; n represents a or g or c or t; y represents c or t; h means a or c or t; v means a or g or c.
[0048] In addition, the existing RT-PCR and Ne...
Embodiment 2
[0055] Specificity and the degree of coverage of the artificial sample evaluation primer of embodiment 2
[0056] Experimental Reagents and Materials
[0057] (1) The new and old primers shown in Table 1 were synthesized by Shanghai Sangon Biotechnology Co., Ltd.
[0058] (2) Sample preparation:
[0059] The oysters selected in this experiment were purchased from Shanghai Luchaogang Aquatic Products Market. Fresh oysters were transported to the laboratory on ice and immediately dissected in a biological safety cabinet. The digestive glands of oysters were divided into 50 mg tubes and stored in RNA keeper (Nova like) until use.
[0060] Stool samples were provided by the Chinese Center for Disease Control and Prevention; DNA molecular rulers and agarose were purchased from Tiangen Biochemical (Beijing) Technology Co., Ltd.; Mofei one-step reverse transcription RT-qPCR kit was purchased from Invitrogen (Shanghai) Trading Co., Ltd.; one-step reverse transcription RT-PCR kit wa...
Embodiment 3
[0074] Example 3 Practical Application of Primers for Evaluation of Actual Oyster Samples
[0075] 156 fresh oysters were collected from Shanghai Luchaogang Aquatic Products Market, transported on ice to the laboratory and immediately dissected in a biosafety cabinet. The digestive glands of the oysters were divided into 50 mg tubes and stored in RNA keeper (Novizyme) until use.
[0076] The total RNA in the oyster sample was extracted according to the instructions of the animal tissue total RNA extraction kit provided by Jerry Biological Co., Ltd., and the extracted RNA was amplified by PCR in a method combining a one-step reverse transcription PCR kit and nested PCR. The PCR product was electrophoresed on a 2% agarose gel for GelRed staining observation, and the gel images of the two were compared for the presence of the target band and the positive rate of GII norovirus.
[0077] The samples with positive test results were tapped to recover the target bands and sequenced, ...
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