HPV detection kit
A detection kit, the technology of the kit, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, microorganisms, etc., can solve the problem that clinical diagnosis cannot be widely used, the detection cost is as high as 500 yuan, and the screening cannot be widely used. and other problems, to achieve the effect of low typing technology cost, reduced inspection cost and high specificity
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Embodiment 1
[0049] Example 1: Primer design.
[0050] Use Vector NTi bioinformatics software to systematically analyze the biological information of various (high-risk and low-risk) HPV genomes, and select the conserved HPV L1 gene fragments in the HPV genome that are not homologous to human and other microbial genome DNA sequences as targets. Primers capable of specifically amplifying all types of HPV L1 gene fragments are designed, and the sequences of the obtained primers are shown in SEQ ID NO: 1-2.
[0051] SEQ ID NO: 1 (F1): 5'---GCCATTAGGTGTGGGCATTAGTGG---3';
[0052] SEQ ID NO: 2 (R1): 5'---TCCTTTGCCCCAGTGTTCCCC---3'.
[0053] The conserved sequences of E6 genes of HPV 16, 31, 33, 35, 51, 58, 59, and 82 types were analyzed by Vector NTi bioinformatics software, and a design that can specifically amplify 16, 31, 33, 35, Primers for 51, 58, 59, and 82 types of high-risk HPV, and the obtained primer sequences are shown in SEQ ID NO: 3-4.
[0054] SEQ ID NO: 3 (F2): 5'---CCGTTGTGTG...
Embodiment 2
[0075] Example 2: Experimental design for HPV detection using the primers obtained in Example 1.
[0076] In this example, HPV molecular detection is completed by 8-tube qPCR. The specific experimental design is as follows: figure 2 shown. Tube 1 is used to detect all types of HPV, including various high-risk and low-risk types; tubes 2 to 7 detect all types of high-risk HPV; tube 8 is the quality control reaction of the β-actin gene internal reference, and the specific functions of tubes 1 to 8 are analyzed as follows :
[0077] (1) Tube 1 uses the primer pair of SEQ ID NO: 1-2. If the reaction of tube 1 is positive, it means that the patient has HPV virus infection, which may be high-risk or low-risk HPV type.
[0078] (2) Tube 2 uses the primer pair of SEQ ID NO: 3-4. If the reaction of tube 2 is positive, it means that the patient has high-risk HPV virus infection, which may be 16, 31, 33, 35, 51, 58, 59 or 82 types; Type 16 accounts for more than 60% of all high-risk ...
Embodiment 3
[0085] Embodiment 3: establishment of standard curve.
[0086] In order to test whether the designed primers can be accurately applied to the qPCR molecular detection of HPV, the siHA cell line known to contain HPV16 was used to detect the standard curve of the primer pair of SEQ ID NO:3-4. A Hela cell line known to contain HPV18 was used to detect the standard curve of the primer pair of SEQ ID NO:5-6.
[0087] The siHA containing HPV16 type and the Hela (cell genomic DNA) containing HPV18 type were respectively extracted with a genomic DNA extraction kit (Kangwei Century Biotechnology, Beijing), and then the genomic DNA was diluted to 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , and 10 7 individual / group. Then use tubes 2 and 3 primer pairs to detect them respectively according to the qPCR reaction system and reaction conditions in Table 1 and Table 2. The results showed that the qPCR CT value was negatively correlated with the amount of genomic DNA, and the reproducibility was...
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