High-precision nucleic acid quantitative detection kit for hepatitis C virus (HCV)

A hepatitis C virus, detection kit technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of poor accuracy, low sensitivity, large sample size, etc., to eliminate interference and achieve good repeatability , a strong specific effect

Active Publication Date: 2014-03-19
东北制药集团辽宁生物医药有限公司
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Problems solved by technology

[0006] The purpose of the present invention is to provide a method for the quantitative detection of hepatitis C virus (HCV) nucleic acid with high precision and its kit, which fundamentally solves the low sensitivity of the existing quantitative PCR technology of hepatitis C virus (HCV) , poor accuracy, etc., the designed primers and TaqMan fluorescent probes are located in the 5'UTR region of the HCV genome. The primers and probes have a wide range of genotype coverage, h

Method used

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  • High-precision nucleic acid quantitative detection kit for hepatitis C virus (HCV)
  • High-precision nucleic acid quantitative detection kit for hepatitis C virus (HCV)
  • High-precision nucleic acid quantitative detection kit for hepatitis C virus (HCV)

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Embodiment 1

[0025] Examples of fluorescence quantitative PCR detection of HCV RNA

[0026] (1) Reagent preparation

[0027] a. Preparation of RT-PCR reaction solution

[0028] The RT-PCR buffer composition is 50mmol / L Tris-HCl (PH8.3), 10mmol / L (NH4) 2 SO 4 , 75mmol / LKCl, 2.5mmol / L MgSO 4 , 0.15mmol / L dNTPs. The upstream and downstream primers used to amplify the target gene were used at a concentration of 20 pmol, the target gene detection probe used at a concentration of 8 pmol, the internal standard upstream primer and downstream primer used at a concentration of 8 pmol, and the internal standard probe used at a concentration of 8 pmol.

[0029] b. Take the corresponding amount of lysis solution binding solution and internal standard according to the proportion (lysis binding solution 400μl / person + internal standard 0.1μl / person portion), mix thoroughly to form the lysis binding solution-mix, and then centrifuge for use.

[0030] c. According to the amount of test sample, negative control, pos...

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Abstract

The invention relates to a high-precision nucleic acid quantitative detection kit for hepatitis C virus (HCV) applied to the field of biomedical clinical diagnosis. The kit comprises a nucleic acid extraction kit based on a paramagnetic particle method and an HCV nucleic acid amplification box, wherein the nucleic acid extraction kit based on the paramagnetic particle method comprises a splitting combining liquid, a rinsing liquid A, a rinsing liquid B, a rinsing liquid C, an eluting liquid and a magnetic bead liquid. The HCV nucleic acid amplification box comprises an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) reaction liquid, enzyme mixed liquor, an HCV-interior label, an HCV quantitative reference 1-4, a negative quality control product, a critical positive quality control product and a positive quality control product. The splitting combining liquid in the nucleic acid extraction agent based on the paramagnetic particle method comprises 0.5-2.5% of lauryl sodium sulfate, 0.5-2.0ml/100ml of TritonX-100, 2-6mol/L of guanidinium isothiocyanate and 1-10mM of EDTA (Ethylene Diamine Tetraacetic Acid) (PH 7.5). The kit provided by the invention is simple and fast to operate, low in cost, high in coverage of primer and probe genotype, high in conservative property, strong in specificity, high in detection sensitivity and good in repeatability, and can better simulate the extraction state of real viruses.

Description

Technical field [0001] The invention relates to a hepatitis C virus (HCV) high-precision nucleic acid quantitative detection kit in the field of biomedical clinical diagnosis. Background technique [0002] Hepatitis C is one of the most serious public health problems in the world, and hepatitis C has a worldwide distribution. The infection rate of Hepatitis C Virus (HCV) is 0.1%-10%, with an average of 3%. About 170-200 million people worldwide are infected with HCV. China has the most chronic hepatitis C (HCV) infections in the world, about 38 million. The chance of hepatitis C virus infection developing into chronic infection is much higher than that of hepatitis B virus infection. Generally speaking, 70-80% of HCV infected persons develop chronic hepatitis C, and the manifestations of chronic hepatitis C vary from severity to severity. 20-30% can gradually develop into liver cirrhosis, and about 1% to 4% of patients with liver cirrhosis can develop into liver cancer each yea...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2545/114C12Q2545/101C12Q2561/101
Inventor 李望丰关尔鑫蔡一荣赵世源王丹肖樊周凯任旭
Owner 东北制药集团辽宁生物医药有限公司
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