Human papillomavirus (HPV) primer and molecular beacon combination based on isothermal amplification, kit and detecting method

A molecular beacon and kit technology, applied in the biological field, can solve problems such as being unsuitable for real-time detection of HPV, and achieve the effects of high sensitivity, fast use and good accuracy

Inactive Publication Date: 2019-10-29
中生方政生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned patents have high requirements for instruments and strict temperature control, and are not suitable for point-of-care testing (POCT) of HPV.

Method used

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  • Human papillomavirus (HPV) primer and molecular beacon combination based on isothermal amplification, kit and detecting method
  • Human papillomavirus (HPV) primer and molecular beacon combination based on isothermal amplification, kit and detecting method
  • Human papillomavirus (HPV) primer and molecular beacon combination based on isothermal amplification, kit and detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Design of primers and molecular beacon combinations for rapid detection of 15 medium and high-risk HPV viruses

[0069] Select the HPV virus gene sequence in NCBI, use Vector NTI software to perform sequence comparison, select the most conserved L1 region, and design primer pairs and molecular beacon combinations. The sequence is as follows:

[0070] Primer 1: 5'-CCAGATCCTAATAAGTTTGGATTTCC-3' (SEQ ID No.1);

[0071] Primer 2: 5'-CCTGATCCCAATAAATTTGGCCTTCC-3' (SEQ ID No.2);

[0072] Primer 3: 5'-CCCGATCCTAATAAATTCAGWNTTCC-3' (SEQ ID No.3);

[0073] Primer 4: 5'-CCTGACCCAAATAAATTTGGWTTACC-3' (SEQ ID No.4);

[0074] Primer 5: 5'-CCTATAGTGAGTCGTATTAAGCGAAATTAATACGACTCACTATAGGCCTAATGGCTGWCCACGGCC-3' (SEQ ID No.5);

[0075] Primer 6: 5'-CCTATAGTGAGTCGTATTAAGCGAAATTAATACGACTCACTATAGGCCCAATGGCTGGCCCCTGCC-3' (SEQ ID No.6);

[0076] Primer 7: 5'-CCTATAGTGAGTCGTATTAAGCGAAATTAATACGACTCACTATAGGCCTAAAGGCTGTCCCCTACC-3' (SEQ ID No.7);

[0077] Molecular beacon 1: 5'-FAM...

Embodiment 2

[0087] Example 2: Establishment of a constant temperature detection kit for rapid detection of 15 medium and high-risk HPV viruses

[0088] A constant temperature detection kit for rapid detection of HPV virus, including reaction mixture, enzyme mixture, primer / molecular beacon combination pair, positive control substance, negative control substance, instructions and box.

[0089] Wherein, the reaction mixture contains 30mM Tris-HCl, 20mM KCl, 2mM MgCl 2 , 20 mM ammonium sulfate, 2 mM dNTPs, 2 mM rNTPs.

[0090] The enzyme mixture contains Bst DNA polymerase, T7 RNA polymerase, RNaseH and UDG enzyme.

[0091] The combination of primers and molecular beacons is as shown in Example 1, the concentration of each primer is 5 μM, and the concentration of each molecular beacon is 2 μM.

[0092] The positive control is a plasmid solution containing a specific target DNA fragment in the L1 region of the HPV virus, and the specific target sequence (SEQ ID No.18) is as follows:

[009...

Embodiment 3

[0095] Embodiment 3: the rapid detection method of 15 kinds of medium and high risk HPV viruses

[0096] Utilize the kit of embodiment 2 to detect HPV virus rapidly, concrete steps are as follows:

[0097] (1) Nucleic acid extraction: use the virus DNA extraction kit to extract the virus DNA in the sample;

[0098] (2) Constant temperature amplification: use the kit of Example 2 to amplify, use the extracted DNA of the sample to be tested, the positive control and the negative control as templates respectively, and use the reaction mixture, enzyme mixture, and primer molecular information of the kit as templates. The standard combination is mixed, and the constant temperature amplification is carried out to detect the fluorescent signal;

[0099] (3) Judgment of results: ① positive control Ct value (threshold cycle) is between 12 and 30, negative control Ct ≥ 65, follow-up judgment should be carried out, otherwise the problem should be searched; ② Ct value of the amplificatio...

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Abstract

The invention belongs to the technical field of biology, and discloses a primer and molecular beacon combination for quickly detecting human papillomaviruses (HPVs) based on an isothermal amplification technology, a kit and a detecting method. The primer and molecular beacon combination is shown as SEQ ID No.1-17, and can specifically recognize the 13 different types of high-risk HPVs and two types of medium-risk genotypes with the high incidence in Chinese population; and according to the kit and the method for real-timely detecting the HPVs through fluorescence based on the isothermal amplification technology, the 15 types of medium-and-high-risk HPVs can be detected within the short time (1.5 h), accuracy is good, sensitivity is high, repeatability is high, specificity is good, no nonspecific reaction with clinically similar pathogens exists, using is convenient, only an extracted template sample needs to be added into a reaction tube, the manual operation time is less than 5 min, the requirement of isothermal amplification for temperature control over an instrument is not high, and thus the primer and molecular beacon combination, the kit and the detecting method can be appliedto instant detection well.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the detection of human papillomavirus pathogenic microorganisms, in particular to a combination of primers and molecular beacons, a kit and a detection method for rapid detection of human papillomavirus based on constant temperature amplification technology. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) belongs to the Papillomaviridae family, is a small molecule non-encapsulated circular double-stranded DNA virus, the genome is about 8000 base pairs (bp), divided into three functions Regions, namely early transcribed region (E region), late transcribed region (L region) and non-transcribed region (long control region, LCR). HPV infects humans through direct or indirect contact with contaminated objects or through sexual transmission. The virus is not only host-specific but also tissue-specific, and can only infect human skin and mucosal epithelial ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12N15/11C12R1/93
CPCC12Q1/708C12Q1/6844C12Q2563/107
Inventor 蔺皓邹国宝魏颖颖宋高尚刘欣欣周保强
Owner 中生方政生物技术股份有限公司
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