Hepatitis C virus genotyping method based on loop-mediated isothermal amplification

A technology of hepatitis C virus and genotyping method, which is applied in the direction of biochemical equipment and methods, microorganism measurement/inspection, etc. It can solve the problems of high laboratory pollution risk, easy amplification failure, high equipment requirements, etc., and achieve low Cost, High Sensitivity, Effect of High Sensitivity

Inactive Publication Date: 2018-12-28
FIRST AFFILIATED HOSPITAL OF KUNMING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

HCV whole genome sequencing is the most accurate method, but the operation is complicated, the sensitivity is low, and the equipment requirements are high
The type-specific primer amplification method of the traditional PCR method is also affected by the primer and has the disadvantage of easy amplification failure.
The RELP method has a high risk of laboratory contamination due to the need to open the lid during the detection process
Therefore, these methods have certain deficiencies in clinical application.

Method used

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  • Hepatitis C virus genotyping method based on loop-mediated isothermal amplification
  • Hepatitis C virus genotyping method based on loop-mediated isothermal amplification
  • Hepatitis C virus genotyping method based on loop-mediated isothermal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] A hepatitis C virus genotyping method, based on the LAMP method, downloads the sequences of HCV subtypes from GeneBank (CHINA HCV 1b:L02836.1, HQ912959.1, HQ912956.1, GU451224.1, HCV 2a:HQ639945. 1, KC844043.1, HQ639944.1, HQ639938.1, KF676352.1, HCV 3: AP008209.2, HQ912953.1, KC844041.1, KC844044.1, HCV 6a: DQ480524.1, HQ912955.195, HQ9 1, KC844037.1, DQ480516.1), using Geneious10.2.3 software to compare the sequences of each subtype, select the specific and conserved sequence segment of each subtype in the 5'UTR-Core region, and use primer explorer V.5 (http: / / primerexplorer.jp / e / ) software to design various types of LAMP reaction primers, including two outer primers (F3, B3), two inner primers (FIP, BIP) and loop primers (LF or LB); Primers were synthesized by Sangon Bioengineering Co., Ltd. (Shanghai, China).

[0052] The reaction system includes: 12.5μL 2×MasterMix (6mM MgSO4, 1.4mM dNTP Mix, 320U Bst3.0DNA Polymerase), 2.5μL primer mixture (40pmol each for FIP an...

Embodiment 2

[0054] A method for genotyping hepatitis C virus 1b, 2a, 3 and 6a comprising:

[0055] Serum samples: A total of 108 serum samples were collected from the First Affiliated Hospital of Kunming Medical University (Yunnan, China) and Kunming Infectious Disease Hospital (Yunnan, China) from August to December 2017 after clinical testing. Among them, 80 specimens were identified by gene sequencing method to determine the subtype of HCV, and the markers of hepatitis A, hepatitis B, HIV and cytomegalovirus were negative, which were used to evaluate the specificity of the method; 10 cases of hepatitis B virus serum, 10 cases of HIV virus serum, 8 cases of hepatitis A virus, and negative hepatitis C markers, used to verify the anti-interference of RT-LAMP method.

[0056] Nucleic acid extraction: Serum samples were extracted using a DNA / RNA magnetic bead method nucleic acid extraction kit (Tianlong Technology Co., Ltd., Xi'an, China), and the nucleic acid extraction method was operated...

Embodiment 3

[0068] Hepatitis C virus genotyping method based on embodiment 2

[0069] Evaluation of the lowest detection limit: HCV1b, 2a, 3, 6a type RNA (concentration is about 1.0×10 6 IU / mL) specimens were diluted with 10-fold gradient with HCV-negative serum to obtain a series of samples (1.0 × 10 6 ,1.0×10 5 ,1.0×10 4 ,1.5×10 3 ,1.0×10 3 ,1.0×10 2 IU / mL), and then divide the samples of each concentration into 10 parts to extract nucleic acid respectively. The above samples were tested by both LAMP and Real-time PCR methods at the same time, and the lowest concentration of all 10 samples positive was regarded as the lowest detection limit.

[0070]The lowest detection limit of HCV-1b, 2a 3 and 6a by LAMP method and Real-time PCR.

[0071] Such as figure 2 as shown ( figure 2 : Evaluation of the lowest detection limit of HCV-1b, 2a, 3 and 6a by LAMP method. A: LAMP method for detection of HCV-1b, 2a, 3 and 6a reaction chart, 1-10 represents 10 repeated reaction wells of the...

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Abstract

The invention relates to a hepatitis C virus genotyping method based on loop-mediated isothermal amplification, and relates to the field of molecular biotechnology. The hepatitis C virus genotyping method is characterized in that different subtypes of LAMP primers are designed based on the LAMP method specific to the specific gene sequences of the subtypes in the 5'UTR-Core region of the hepatitisC virus, including two outer primers, two inner primers and a loop primer, and an NEB reagent is adopted for detection at 65 DEG C for 60 min. The method has high specificity, high sensitivity and low cost, can achieve high-throughput automatic detection and contamination risks of specimens. Low equipment requirements can help promote the clinical application of the LAMP method. The method has good clinical application prospects. At the same time, the development of corresponding reagents is conducive to further promoting the clinical application of the method.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, in particular to a method for genotyping hepatitis C virus based on loop-mediated constant temperature amplification (Loop-mediated isothermal amplification, LAMP). Background technique [0002] Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus belonging to the genus Hepatitis C in the family Flaviviridae. There are about 170 million people infected with hepatitis C virus in the world, and the infection rate of HCV in China accounts for 3.2% of the total population, about 42 million. HCV infection develops insidiously and has a relatively high rate of chronicity. More than 80% of them will turn into chronic hepatitis after infection. Without reasonable treatment, 10% to 30% of them will develop into liver cirrhosis after 10 to 20 years, and 1% will develop into liver cirrhosis. ~3% develop primary liver cancer [3-5] . The HCV genome is highly heterogeneous...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844
CPCC12Q1/6844C12Q1/707C12Q2531/119
Inventor 刘子杰段勇杨川琪
Owner FIRST AFFILIATED HOSPITAL OF KUNMING MEDICAL UNIV
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