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Multiplex PCR primer group, kit and method for detecting HPV based on high-throughput sequencing

A high-throughput, primer set technology, applied in the field of next-generation sequencing, can solve the problems of inaccurate detection results, easy contamination of samples, large sample volume, etc., and achieve the effects of strong specificity, wide coverage, and prevention of interference

Active Publication Date: 2020-04-17
广州迈景基因医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to provide a HPV detection method based on high-throughput sequencing and its application, using multiple PCR methods to solve the problems of easy sample contamination, inaccurate detection results, and large sample size during the sample detection process.

Method used

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  • Multiplex PCR primer group, kit and method for detecting HPV based on high-throughput sequencing
  • Multiplex PCR primer group, kit and method for detecting HPV based on high-throughput sequencing
  • Multiplex PCR primer group, kit and method for detecting HPV based on high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Primer Design

[0044] Primer design, including multiple PCR primers and linker primers.

[0045] Multiplex PCR primers contain specific and bridging sequences. The specific sequence is designed for 40 types of HPV, all of which are degenerate primers, and the annealing temperature is controlled at 58°C to 62°C; the bridging sequence is the same part of all upstream primers or downstream primers, mainly combining with the bridging sequence of the linker primer part, adjust the length of the bridging sequence according to the actual situation.

[0046] From the 5' end to the 3' end of the linker primers are: the general sequence related to the sequencer, the 8bp sample index sequence, and the bridge sequence matching the 5' end of the multiplex PCR primers. The sample labels of each adapter primer band are different, and the annealing temperature of the bridging sequence part is between 68°C and 72°C. The 8bp sample label in the adapter primer can be replace...

Embodiment 2

[0057] Embodiment 2 artificially synthesized plasmid specificity and sensitivity test

[0058] Artificially synthesized 7 plasmids: HPV16, HPV18, HPV31, HPV33, HPV35, HPV58, HPV68. Three test concentrations were set up, each of which was 10 5 copy / test, 10 3 copy / test, 10 2 Copy / test. Set three positive controls (both HPV16, HPV18, HPV31, HPV33, HPV58, HPV35, the mixture of plasmids such as HPV68 and human genomic DNA: PC-1 (200 copies / test), PC-2 (1000 copies / test), PC-3 (5000 copies / test), and a negative control (NC), a total of 25 samples, adapter primer settings are shown in Table 1:

[0059] Table 1 Adapter primer settings

[0060]

[0061]

[0062] Preparation of PCR amplification reaction system:

[0063] Reagent Final concentration PCR Mix 1× Primer library 0.5μM Any P5 connector 0.4μM Any P7 connector 0.4μM dna Make up 20μL

[0064] PCR amplification reaction program settings:

[0065]

[0066] Build t...

Embodiment 3

[0076] Example 3 Clinical Sample Consistency Test

[0077] 92 cases of cervical swab samples from clinical sources were tested positive for HPV by fluorescent PCR method, and genomic DNA was extracted with QIAamp DNAblood mini kit. One test per sample, a total of 92 tests, marked as sample 1 to sample 92; Set three positive controls PC-1 (200 copies / test), PC-2 (1000 copies / test), PC-3 (5000 copies / test), and a negative control (NC), a total of 96 samples. Complete the test of a 96-well plate, and the adapter primer settings are shown in Table 3:

[0078] Table 3 Adapter primer settings

[0079]

[0080] Preparation of PCR amplification reaction system:

[0081] Reagent Final concentration PCR Mix 1× Primer library 0.5μM Any P5 connector 0.4μM Any P7 connector 0.4μM dna Make up 20μL

[0082] PCR amplification reaction program settings:

[0083]

[0084] Build the library:

[0085] 1) After the PCR reaction is complet...

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Abstract

The invention discloses a multiplex PCR primer group and a kit for detecting HPV based on high-throughput sequencing. Existing methods are difficult to realize HPV high-throughput sample detection. When a large amount of samples need to be detected, a large amount of manpower is needed for operation. According to a method established by the invention, amplification and purification can be performed through a one-step method, a library is established through the one-step method, tube transform and cover opening are avoided in the midway, and confusion or cross contamination among samples is effectively avoided; library construction of 96 samples can be completed within 5 hours, operation is easy and convenient, and detection of a large number of samples can be easily achieved. By increasingthe number of sample tags in an index primer, the number of samples which can be detected through one-time sequencing can be increased, and the average manual operation time of a single sample is further shortened.

Description

technical field [0001] The invention belongs to the field of next-generation sequencing, and in particular relates to a multiplex PCR primer set, a kit and a method for detecting HPV based on high-throughput sequencing. Background technique [0002] According to the statistics of the World Health Organization in 2018, cervical cancer is the fourth largest female malignant tumor in the world. In 2018, there were nearly 570,000 new cases and more than 310,000 deaths due to cervical cancer. Globally, the number of cervical cancer patients is increasing year by year and getting younger. The number of new cases in my country exceeds 100,000 each year; the number of cases in other developed countries / regions exceeds 85% of the total. [0003] Cervical cancer is currently the only malignant tumor with a clear etiology that can be prevented and cured early. 99.9% of cervical cancers are related to human papillomavirus (Human Papilloma Virus; HPV) infection. Persistent infection of ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6869C12N15/11C12R1/93
CPCC12Q1/6869C12Q1/708C12Q2600/16C12Q2531/113C12Q2537/143C12Q2535/122
Inventor 蔡兴盛李梦真邓泱泱王春林马志海钱冬媛刘雅思乐小炎杨冬成
Owner 广州迈景基因医学科技有限公司
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