Reagent and method for qualitatively detecting BoHV1
A bovine herpes virus, type I technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome operation process and expensive, and achieve simple operation, fast detection and saving The effect of instrument cost
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Embodiment 1
[0071] Example 1: Primer Design
[0072] The inventors designed and screened a set of specific LAMP primers with the gC gene-specific site of BoHV1 as the target gene. The sequences of these primer sets are:
[0073] BoHV1-F3: CAGCTTGAGCTTCTCCACG (SEQ ID NO: 1);
[0074] BoHV1-B3: CTGGGCGTACATCTCGGA (SEQ ID NO: 2);
[0075] BoHV1-FIP: GCCAAAACCGCTTTCAGAAGCACATGTCCAGGGAAACCACG (SEQ ID NO: 3);
[0076] BoHV1-BIP: GCGTCGAAGACGCGGAAGACTCACGGCCGTCTACGA (SEQ ID NO: 4).
[0077] In addition, on the basis of the above sequence, the inventors also introduced a pair of loop primers to increase the amplification efficiency, the sequence of which is:
[0078] BoHV1-LF: GAGCAAGGTGAAGATTAACGG (SEQ ID NO: 5);
[0079] BoHV1-LB: TTAGCTTCCCGTAGCCGTC (SEQ ID NO: 6).
[0080] By using BLAST software for comparison, the inventors found that the sequences of the above six primers matched the corresponding sequences of the BoHV-1 virus in the database, and had no obvious crossover with other v...
Embodiment 2
[0081] Example 2: Detection of BoHV1
[0082] This embodiment provides exemplary specific steps for detecting BoHV1 on the sample to be tested.
[0083] First, the virus DNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract DNA.
[0084] Then, prepare the reagents. See Table 1 for the stock solution concentrations of the reagents used in the LAMP amplification reaction. In the case of N samples to be tested, refer to Table 1, and calculate the dosage of each component according to the number of N+3 (N samples to be tested+1 negative control+1 positive control, prepare one more reaction to ensure that the reaction solution Accurate dispensing volume), add each reaction reagent into a 1.5mL PCR centrifuge tube, vortex or cover the lid and mix upside down 10-20 times, centrifuge for 30 seconds, dispense 23μL into each PCR tube, and pour into Add 1 drop of sealing solution (approximately 20 μL) to each tube.
[0085] Table 1...
Embodiment 3
[0089] Embodiment 3: Optimization of LAMP reaction system
[0090] Usually, the temperature of the LAMP amplification reaction is kept at 60° C. to 67° C., and the reaction time is 40 to 60 minutes. The present inventors found that the preferred reaction temperature for LAMP detection using the primer set of the present invention is 63° C., and the preferred reaction time is 45 minutes.
[0091] On this basis, the inventors also found that, in addition to reaction temperature and reaction time, dNTP and Mg 2+ The concentration also plays an important role in the detection effect. In order to obtain the best detection effect, the present invention targets different dNTPs and Mg 2+ Concentrations were tested in a series, as shown in Table 2 below. In addition, 6 specific primers were used in this example.
[0092] Table 2 2: dNTPs and Mg in optimization tests 2+ concentration
[0093] experiment number MgSO 4
[0094] Test results such as figure 2 As shown,...
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