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Nucleotide sequence and kit for detecting hepatitis C viruses

A hepatitis C virus, nucleic acid sequence technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem that the performance indicators cannot reach clinical diagnosis and treatment, and the detection sensitivity and stability are poor. , unable to meet the detection needs and other problems, to achieve the effect of high specificity, high sensitivity and high accuracy

Inactive Publication Date: 2018-02-02
HANGZHOU D A GENETIC ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The primers and probes designed by existing quantitative reagents are often based on the type and sequence information of HCV infection several years ago, which cannot meet the detection requirements of emerging subtypes, resulting in a decrease in detection sensitivity, poor stability, and performance indicators that cannot meet clinical requirements. Diagnosis and treatment requirements, difficult to meet the latest clinical needs for diagnosis and treatment (below 30IU / mL)
And the hepatitis C ultrasensitive detection kit with the best performance on the existing market is the Swiss company Roche AmpliPrep / HCV Test, v2.0, although it can reach 30IU / mL, it also uses PCR-fluorescence method, which also cannot avoid the problem of poor detection sensitivity and stability near the detection limit

Method used

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  • Nucleotide sequence and kit for detecting hepatitis C viruses
  • Nucleotide sequence and kit for detecting hepatitis C viruses
  • Nucleotide sequence and kit for detecting hepatitis C viruses

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: The primers and probes that detect HCV RNA and human internal reference gene ERG are synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd., and the sequences are as follows:

[0037] Primers for amplifying HCV DNA:

[0038] F1 (SEQ ID NO: 1): 5'-ccgggagrgccatagtrgtct-3';

[0039] R1 (SEQ ID NO: 2): 5'-gtttatccaagaaaggacccdgtc-3';

[0040] Probes to detect HCV RNA:

[0041] PHCV (SEQ ID NO: 3): 5'-actcaccggttccg-3';

[0042] Wherein, r represents a degenerate base (a or g), and d represents a degenerate base (g or a or t); the 5' end of the HCV probe PHCV (SEQ ID NO: 3) sequence is marked with a FAM fluorescent group, The 3' end is labeled with the MGB quencher group,

[0043] Primers for amplifying the human ERG gene:

[0044] F3 (SEQ ID NO: 4): 5'-cagaacacagcagagggaagg-3';

[0045] R3 (SEQ ID NO: 5): 5'-aagctcccacttccataaaggc-3';

[0046] Probes for detection of human ERG gene:

[0047] PERG (SEQ ID NO: 6): 5'-aggagtcccaggctc-3';

[0048] Whe...

Embodiment 2

[0049] Example 2: Preparation method of HCV RNA quantitative kit.

[0050] (1) PCR reaction solution: 4*supermix (purchased from U.S. Bio-rad company, article number 1863026), is 4*PCR reaction premix, wherein contains PCR reaction buffer solution of the present invention, hot-start DNA polymerase, Mg 2+ and dNTPs and other components, stored at -20°C;

[0051] (2) Reverse Transcriltase: Store at -20°C

[0052] (3) DTT: 300nM: Store at -20°C

[0053] (4) Primer-probe premix solution: Submit the nucleotide sequences shown in SEQ ID NO: 1 to 6 to Yingwei Jieji (Shanghai) Trading Co., Ltd. for synthesis. After getting the dry powder, use deionized water to separate the Dilute 4 primers to a concentration of 100uM, dilute two probes to a concentration of 10uM, then mix the 6 primer-probe master solutions at a ratio of 9:9:25:9:9:25, and store at -20°C;

[0054] (5) Positive control: Serum samples containing HCV 7 genotype plasmids (purchased from the China Institute of Standard...

Embodiment 3

[0057] Embodiment 3: detection method.

[0058] Instruments: Eppendorf Mastercycler pro S qualitative PCR instrument, Bio-rad QX200 droplet digital PCR system (including droplet generator, sealing device, droplet reader), BECKMAN 22R desktop micro-refrigerated centrifuge, WH-866 vortex oscillator (Taicang Hualida), low-speed plate centrifuge (Anhui Zhongjia).

[0059] (1) Preparation of HCV patient samples, positive control, and negative control RNA templates: Use QIAGEN Serum Virus Extraction Kit (column method, Cat. No. 57704), operate according to the kit instructions, and extract patient samples into 500 μL serum in advance Add 2.5 μL of the internal reference plasmid, and then extract; take 500 μL of negative and positive control samples for extraction directly, and use 50 μL of eluent for elution, and use it as a PCR reaction template for later use.

[0060] (2) using the nucleic acid obtained in step (1) as a template, using 3 pairs of specific primers and two specifi...

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Abstract

The invention discloses a nucleotide sequence and a kit for detecting hepatitis C viruses (HCV), and belongs to the technical field of molecular diagnosis. By a primer and a probe for detecting the HCV, virus RNA of seven HCV genotypes can be specifically amplified and detected. By the kit for detecting the hepatitis C viruses, accurate copy numbers of various subtypes of the HCV can be detected quickly, sensitively and accurately. The nucleotide sequence and the kit have the advantages of simplicity and convenience in operation, high specificity, high sensitivity, low cost, high throughput and the like, can be applied to clinical quick hyper-sensitive detection of HCV virus RNA, and provide reference to clinical diagnosis and treatment of hepatitis C.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a nucleic acid sequence and a kit for detecting hepatitis C virus. Background technique [0002] Hepatitis C is a kind of viral hepatitis caused by hepatitis C virus (Hepatitis virus C, HCV) infection, and hepatitis C virus is the source of morbidity and infection of hepatitis C patients. Hepatitis C is one of the most serious public health problems in the world. Hepatitis C is distributed worldwide. China has the largest number of chronic hepatitis C virus (HCV) infections in the world, about 38 million. HCV infection is much more likely to develop into a chronic infection than HBV infection. Generally speaking, 70-80% of HCV-infected patients develop chronic hepatitis C. The severity of chronic hepatitis C varies, and about 20-30% can gradually develop into liver cirrhosis. Among patients with liver cirrhosis, about 1-4% per year Can develop into liver...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/707C12Q1/6851C12Q2600/166C12Q2563/159C12Q2537/16C12Q2563/107
Inventor 任绪义虞闰六罗英宣文静
Owner HANGZHOU D A GENETIC ENG
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