Kit for human papilloma virus E6/E7 gene detection and detection method
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A human papillomavirus and gene detection technology, applied in the fields of life sciences and methods, can solve the problems of decreased sensitivity of detection of HPV, deletion of HPVL1 gene, and difficulty in grasping the amount of sample DNA.
Active Publication Date: 2016-08-24
SUZHOU MUNICIPAL HOSPITAL
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But, the present invention has following weak points: (1) because the process that HPV integrates into people's genome easily causes HPV L1 gene deletion; This method selects HPV L1 gene as target sequence and may cause the sensitivity of detecting HPV to descend
(2) Since the L1 gene is the most conserved gene among HPV subtypes, the selection of the HPV L1 gene as the targe
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Embodiment 1
[0131] 1. Sample DNA extraction
[0132] Take 200uL of the cervical exfoliated cell suspension to be tested and put it into a 1.5mL tube, centrifuge at 13000r / min for 5min, discard the supernatant, add 200uL of nucleic acid extraction solution to the precipitate, shake and mix well, lyse at 1000C for 10min, centrifuge at 13000r / min for 5min, take the upper 3uL of the supernatant was used as a template for the PCR reaction.
[0133] 2. Multiplex PCR reaction
[0134] The PCR reaction template is added to the reaction system, the reaction system is 10uL: DDH 2 O 2.35uL, 10*PCR Buffer1uL, 25nM Mg 2+ 1uL, 1.5uL of 2nM DNTP, 1uL of primer mixture (i.e. the primers shown in Table 1, the working concentration of each primer is 0.33uM), 0.15uL of 5U / uL FastTaq enzyme, 3uL of sample DNA (PCR reaction template). PCR cycle program: 95°C 4min; 11 cycles x (94°C 30s, 68°C-0.5°C / cycle 90s); 28cycles x (94°C 30s, 63°C 90s); 72°C 5min; 4°C for ever.
[0135] The multi-cycle reaction prog...
Embodiment 2
[0142] 1. Sample DNA extraction
[0143] Take 200uL of the cervical exfoliated cell suspension to be tested, use the automatic nucleic acid extractor NP-968 nucleic acid automatic extractor to extract DNA according to the instructions, and take 3uL as a PCR reaction template.
[0144] 2. Multiplex PCR reaction
[0145] The PCR reaction template is added to the reaction system, the reaction system is 10uL: DDH 2 O 2.35uL, 10*PCR Buffer1uL, 25nM Mg 2+ 1uL, 1.5uL of 2nM DNTP, 1uL of primer mixture (ie, the primers shown in Table 1, the working concentration of each primer is 0.33uM), 0.15uL of 5U / uL FastTaq enzyme, and 3uL of sample DNA. PCR cycle program: 95°C 4min; 11 cycles x (94°C 30s, 68°C-0.5°C / cycle 90s); 28cycles x (94°C 30s, 63°C 90s); 72°C 5min; 4°C forever.
[0146] 3. Genetic analyzer on PCR products
[0147] Take 1uLPCR product using DDH 2 O was diluted 50 times, and 1uL of the diluted solution was mixed with 0.06μl Liz120SIZESTANDARD and 8.9μl Hi-Di, and put on ...
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Abstract
The invention discloses a kit for human papilloma virus E6/E7 gene detection. The kit comprises 22 pairs of upstream primers and downstream primers designed by aiming at 22 kinds of different subtypes of human papilloma viruses, wherein one of each pair of upstream primer and downstream primer is modified with a fluorophore; each pair of PCR (polymerase chain reaction) primers aims at one kind of HPV E6/E7 gene. The invention also discloses a method for human papilloma virus E6/E7 gene detection. The method comprises the following steps that 10uL of PCR reaction systems, 2.35uL of DDH2O, 1uL of 10*PCR buffer, 1uL of 25nM Mg2<+>, 1.5uL of 2nM DNTP, a mixture of 22 pairs of upstream primers and downstream primers designed by aiming at 22 kinds of different subtypes of human papilloma viruses, 0.15uL of 5U/uL FastTaq enzymes and 3uL of reactive templates are used; a genetic analyzer is used for performing capillary electrophoresis and genotype analysis on a PCR product; the HPV subtypes are judged according to the specific color and position peak on the capillary electrophoresis figure. The method provided by the invention adopts a Touchdown multiplex PCR technology, so that the multiplex PCR non-specificity amplification can be effectively reduced.
Description
technical field [0001] The invention belongs to the field of life science and technology, in particular to the detection of human papillomavirus HPV. Background technique [0002] Persistent infection with human papillomavirus (HPV) is a necessary condition for cervical cancer. Detection of HPV DNA in cervical exfoliated cells has become an important means of screening cervical cancer and precancerous lesions. Various nucleic acid detection technologies have also been applied to HPV DNA detection. There are more than ten kinds of common HPV DNA detection products on the market, which are mainly divided into hybridization method, real-time quantitative PCR method, second-generation hybridization capture method, pyrosequencing method and flight mass spectrometry technology according to the technical principles. According to whether to type HPV, HPV DNA detection technology is divided into two categories: qualitative and quantitative. With the progress of research, it has be...
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