[0032] Example 2. Primers are used to detect HBV by PCR
[0033] 1. Preparation of samples to be tested
[0034] A total of 32 samples of slaughtered chicken liver were collected, labeled and stored at -80°C.
[0035] Put about 100mg of each 4 samples to be tested in a mortar, add liquid nitrogen to grind and mix them, then use a 1.5ml centrifuge tube to collect samples and mix and grind about 100mg of tissue for subsequent tests. There are 8 sets of mixed samples to be tested. The mixed samples of each collection should be marked for the second test.
[0036] One group: numbered 13-16, two group: numbered 17-20, three group: numbered 1-4, four group: numbered 21-24, five group: numbered 25-28, six group: numbered 5-8, seven groups: numbered 9-12, eight groups: numbered 29-32. Among the above 32 samples, samples with codes 1, 5, 8, and 13 are known to be infected with HBV, and the rest are not infected.
[0037] 2. Collecting sample PCR
[0038] 1. Genomic DNA extraction (take Kangwei Century Universal Column DNA Extraction Kit as an example):
[0039] 1) Add 180μl of BufferGTL to each centrifuge tube containing 100mg of mixed sample to be tested, and label it at the same time;
[0040] 2) Add 20μl of proteinase K (Proteinase K), vortex and shake to mix the sample, and bath at 56°C for 1-3 hours until the tissue is completely lysed. If there is too much tissue, increase the proteinase K content or prolong the floret time;
[0041] 3) Add 200μl of BufferGL and vortex to mix. Then add 200μl of pre-cooled absolute ethanol, vortex and mix well;
[0042] 4) After instant centrifugation, transfer all the solution obtained in the previous step to an adsorption column equipped with a collection tube. Centrifuge at 10000rpm/min for 1min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;
[0043] 5) Add 500μl of BufferGW1 to the adsorption column, centrifuge at 10000rpm/min for 1min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;
[0044] 6) Add 500μl of BufferGW2 to the adsorption column, other operations are the same as the previous step, if you need to increase the amount of DNA extraction, this step can be repeated;
[0045] 7) After centrifugation at 12000 rpm/min for 2 minutes, discard the waste liquid in the collection tube, and let the adsorption column stand at room temperature for a few minutes to completely dry;
[0046] 8) Put the adsorption column into a new centrifuge tube, add 50-150μl GE or sterile water to the middle part of the adsorption column, leave it at room temperature for 2-5min, centrifuge at 10000rpm/min for 1min, collect the DNA solution, freeze it at -20℃ for later use , Get the genomic DNA of 8 sets of mixed samples.
[0047] If you need to increase the amount of recovery, this step is also repeated.
[0048] 2. PCR amplification
[0049] Using the genomic DNA of each of the above-mentioned mixed samples as a template, perform PCR amplification with S1-F/S1-R to obtain the first amplification product, and then use the first amplification product as a template, using S2-F/S2 -R performs PCR amplification to obtain secondary PCR products.
[0050] The primer sequences are shown in Table 1, and the PCR amplification system is shown in Table 2.
[0051] Table 2 shows the HBVDNA PCR amplification system
[0052]
[0053] The reaction conditions for the first round of PCR amplification of the HBVS gene were: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 40s, 56°C annealing for 20s, 72°C extension for 30s; after 35 cycles, 72°C extension for 10 minutes.
[0054] The reaction conditions for the second round of PCR amplification of the S gene were: 94°C pre-denaturation for 10 minutes; 94°C denaturation for 20s, 50°C annealing for 20s, 72°C extension for 30s; after 40 cycles, 72°C extension for 7 minutes.
[0055] Prepare a 1.2% agarose gel, take 5μl of secondary PCR product spot, 80V voltage electrophoresis for 30min, take pictures and analyze in the gel imaging system.
[0056] The result is figure 1 As shown, M: DNAMarker2000; 1-8: one to eight mixed samples respectively; 9: negative control, it can be seen that the size of the PCR products obtained in the three, six and seven groups is about 281 bp, and the other mixed samples have no target products .
[0057] Then, each test sample in the third, sixth, and seventh groups is subjected to two PCR amplifications according to the above method, and the second PCR amplification products are detected by electrophoresis.
[0058] If the secondary PCR amplification product of which sample has a fragment of 281bp, the sample to be tested is infected or the candidate is infected with HBV virus;
[0059] If the secondary PCR amplification product of which sample does not have a fragment of 281bp, the sample to be tested is not infected or the candidate is not infected with HBV virus.
[0060] The result is figure 2 As shown, M: DNAMarker2000; 1-4: individual samples numbered 1-4 in the three groups, 5-8: individual samples numbered 5-8 in the six groups, 9: negative control; 10: DNAMarker2000; 11-14: separate samples numbered 9-12 among the seven groups; 15: negative control;
[0061] It can be seen that the samples coded as 1, 5, 8, 13 obtained 281bp secondary PCR amplification products, which were infected with HBV virus; the remaining samples had no PCR products and were not infected; the positive detection rate was 12.5% (4/ 32).
[0062] The secondary PCR amplification products of the samples coded as 1, 5, 8, and 13 were respectively sequenced, and the results were compared with the human HBVs gene (genbank number is KF917521.1, submitted in December 2013), and the results were compared with human The homology of HBV is 97.5%-99.3%; it further proves that the method of the present invention is correct.
[0063]
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