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Reagent for detecting human papillomavirus

A human papillomavirus and reagent technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of unrealistic large-scale application and high experimental technical requirements, and achieves low cost and detection sensitivity. High and accurate effect

Active Publication Date: 2011-01-26
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires expensive instrumentation and is highly technically demanding
[0006] The gold standard method for virus detection is whole genome sequencing, but this is unrealistic for large-scale clinical application

Method used

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  • Reagent for detecting human papillomavirus
  • Reagent for detecting human papillomavirus
  • Reagent for detecting human papillomavirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, the design of primer composition A and primer composition B

[0056] 1. Primer Composition A

[0057] More than 100 types of HPV have been found, and there are about 10% base differences in the sequence between different types. Since the HPVL1 gene sequence is relatively conservative, universal primers can be designed for regions with good consistency to amplify multiple subtypes at the same time. Primer composition A consists of 19 primers listed in Table 1, and each primer is individually packaged. The DNA shown in sequence 1 to sequence 5 is a forward universal primer; the DNA shown in sequence 6 to sequence 18 is a reverse universal primer. The DNA shown in Sequence 19 is a universal primer for sequencing.

[0058] The nucleotide sequence of each primer in the primer composition A of table 1

[0059]

[0060] 2. Primer composition B

[0061] Based on the guiding significance of high-risk HPV infection for clinical diagnosis, 12 pairs of specifi...

Embodiment 2

[0065] Embodiment 2, preparation for detecting the reagent of human papillomavirus

[0066] The reagents for detecting human papillomavirus consist of primer composition A, primer composition B and quality control primer pair designed in Example 1. The quality control primer pair is used to amplify the β-globin gene (housekeeping gene), which consists of the DNA shown in sequence 44 and the DNA shown in sequence 45. Sequence 44 (beta globin forward primer): 5'-CAACTTCATCCACGTTCACC-3'; Sequence 45 (beta globin reverse primer): 5'-GAAGAGCCAAGGACAGGTAC-3'.

Embodiment 3

[0067] The application of the reagent prepared by embodiment 3, embodiment 2

[0068] One. The reagent prepared in Example 2 is used to detect human papillomavirus in the sample to be tested

[0069] The reagents prepared in Example 2 were used to detect the TCT samples (cervical slices of patients with informed consent) of 18 informed consent patients respectively, and the specific steps were as follows:

[0070] 1. Preparation of DNA template

[0071] ①Take 1ml clinical sample and add it to 1.5ml Eppendorf, centrifuge at 8000rpm for 1min, and discard the supernatant.

[0072] ② Add cell eluent (containing 137mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 , 2mM KH 2 PO 4 Phosphate buffer solution, pH=7.4) shake evenly, centrifuge at 8000rpm for 1min, and discard the supernatant.

[0073] ③ Add 100 μl of DNA extraction solution (10 mM Tris-HCl solution, pH=7.4), freeze at -80°C for 4-5 hours.

[0074] ④ Place the centrifuge tube in boiling water and boil for 10 minutes.

[0075...

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Abstract

The invention discloses a reagent for detecting a human papillomavirus (HPV). The reagent provided by the invention comprises a primer composition A and a primer composition B, wherein the primer composition A consists of DNA shown in a sequence table from sequence 1 to sequence 18; and the primer composition B consists of DNA shown in the sequence table from sequence 20 to sequence 43. The reagent can be used for assisting in identifying the HPV. The reagent provided by the invention can detect all the types of the HPV in an infected female genital tract, wherein the HPV comprises 14 high-risk types of HPV and 12 low-risk types of HPV. The reagent mainly solves the technical problem that the infected HPV cannot be typed or the typing is not accurate caused by a cross reaction in the conventional detection method so as to provide an accurate, high-sensitivity, economic and practical detection method which is suitable for clinical HPV detection and large-scale crowd HPV screening.

Description

technical field [0001] The invention relates to a reagent for detecting human papillomavirus. Background technique [0002] According to the survey by the International Organization for Research on Cancer (IARC), cervical cancer is the third most common malignant tumor among women, with about 80,000 new cases and 30,000 deaths in China every year. Studies have shown that human papillomavirus is closely related to the occurrence of cervical cancer, and HPV infection can be detected in more than 99% of cervical cancer patients. More than 100 types of HPV have been found so far, of which more than 40 infect the reproductive tract (de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H.Classification of papillomaviruses.Virology.2004.324: 17 -27.). According to epidemiological investigation and molecular phylogenetic analysis, HPV can be divided into high-risk and low-risk types, among which high-risk HPV includes 15 types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 朱宝利向阳杨毅张瑞芬蔡玉品王志云
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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