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Quantitative detection kit for hepatitis B viral nucleic acid

A hepatitis B virus, detection kit technology, applied in the direction of microbial determination/inspection, microorganisms, biochemical equipment and methods, etc., can solve the problem of long amplification time, achieve high amplification efficiency, easy storage and transportation, The effect of improving convenience

Inactive Publication Date: 2018-12-07
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a quantitative detection kit for hepatitis B virus nucleic acid, in order to fundamentally solve the problem that the amplification reagents of the existing hepatitis B virus (HBV) nucleic acid detection kit need to be stored at -20°C and the amplification time is relatively long technical issues

Method used

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  • Quantitative detection kit for hepatitis B viral nucleic acid
  • Quantitative detection kit for hepatitis B viral nucleic acid
  • Quantitative detection kit for hepatitis B viral nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 The composition of kit of the present invention

[0026] 1. PCR reaction solution 1 20µl, which contains 4 specific primers (SEQ ID No.1~4) and 2 specific probes (SEQ ID No.5~6), the primers of SEQ ID No.1~4 The concentration is 25 pmol / µl, the concentration of probe SEQ ID No.5~6 is 15 pmol / µl; 20mM dNTP, 10×PCR reaction buffer, 10U of bifunctional enzyme, 0.4U of UNG enzyme, 1.1µl of enzyme protection solution.

[0027] 2. PCR reaction solution 2 10µl: contains 25mM MnCl 2 , 0.09% sodium azide.

[0028] 3. DNA internal control: artificially constructed plasmid, the concentration is 1.0E+05-1.0E+06copies / ml.

[0029] 4. Quantitative reference products A, B, C, and D: all are pseudoviruses containing the target fragment of HBV.

[0030] 5. Negative quality control: HBV negative serum or plasma,

[0031] 6. Strong positive quality control and critical positive quality control: a pseudovirus containing the target fragment of HBV.

Embodiment 2

[0032] The detection of embodiment 2 HBV DNA

[0033] 1. Build a reaction system

[0034] Calculate the number of samples required, and increase the amount of reagents in proportion to the amount in the table below. Only one copy of strong positive quality control, critical positive quality control, and negative quality control is made.

[0035]

[0036] 2. On-board inspection

[0037] Directly use the fluorescent quantitative PCR instrument for detection, the amplification program is set to 50°C for 2min, 93°C for 2min, (93°C for 5s, 55°C for 20s) 45Cycle, 40°C for 10s; collect FAM and ROX fluorescence after each cycle.

[0038] 3. Quality control

[0039] Each batch of tests should contain a strong positive control, a borderline positive control, and a negative control, all controls should be unmarked and: Positive controls: 1.0E+05~1.0E+07 IU / mL; critical positive quality control: Ct value should be <45; negative quality control: No Ct in FAM channel, Ct value in RO...

Embodiment 3

[0042] Embodiment 3 The minimum detection limit of the kit of the present invention

[0043] Use nucleic acid-negative plasma to dilute the reference product with the lowest detection amount in the national reference product (code: 300009-201602) to 5IU / ml, and use the nucleic acid extraction and purification reagent (magnetic bead method) of Zhengzhou Antu Bioengineering Co., Ltd. to perform type B Hepatitis virus DNA was extracted and purified, and tested using this kit, repeated 20 times. The test results are shown in Table 1.

[0044] Table 1

[0045]

[0046] It can be seen from Table 1 that the minimum detection limit of this kit can reach 5 IU / ml when 20 repeated tests are performed.

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Abstract

The invention discloses a quantitative detection kit for hepatitis B viral nucleic acid. The kit comprises a PCR reaction liquid 1, a PCR reaction liquid 2, DNA internal control, quantitative reference substances A, B, C and D, a strong positive quality control substance, a clinical positive quality control substance and a negative quality control substance. The PCR reaction liquid 1 contains upstream and downstream primers for amplifying polynucleotide of a target, a probe for detecting polynucleotide of the target, a DNA internal control primer and a probe, a PCR reaction buffer solution, dNTPs and an enzyme mixed solution. The kit is high in sensitivity (the lowest detection limit is 5IU / ml, wide in linear range (can cover 20-1.0E+09IU / ml), high in specificity (a pair of primers is designed for an S region sequence of a conservative fragment and does not interfere with other pathogens in a crossed manner), high in amplifying efficiency (the spatial conformation of double functionalenzyme is stabilized by adding an enzyme protecting solution and the activity is enhanced), quick to detect (the whole detection process is finished within 50 min) and convenient to store and transport and free of freezing and repeated freeze-thawing, so that the using convenience of a user is improved.

Description

technical field [0001] The invention relates to in vitro diagnostic technology, in particular to a hepatitis B virus nucleic acid quantitative detection kit. Background technique [0002] Hepatitis B virus (HBV) is a hepatotropic virus that mainly exists in liver cells and damages liver cells, causing inflammation, necrosis, and fibrosis of liver cells. It is the main cause of human viral hepatitis. HBV infection is prevalent worldwide, but the epidemic intensity of HBV infection varies greatly in different regions. According to the report of the World Health Organization, about 2 billion people in the world have been infected with HBV, of which 240 million people are chronically infected with HBV, and about 650,000 people die of liver failure, cirrhosis and hepatocellular carcinoma (HCC) caused by HBV infection every year. Therefore, the prevention, diagnosis and treatment of hepatitis B virus is the top priority of national infectious disease control. [0003] HBV enzyme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/706C12Q2531/113C12Q2563/107
Inventor 李静静李振红杜美鲁清月付光宇吴学炜苗拥军
Owner AUTOBIO DIAGNOSTICS CO LTD
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