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Multiple PCR detection kit for virus hepatitis pathogens and preparation and application thereof

A detection kit, technology for viral hepatitis, applied in the field of biological sciences, able to solve problems such as limitations

Active Publication Date: 2012-09-05
SHANGHAI BLOOD CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, in the actual application process of multiplex PCR technology, since multiple primers are added in the same reaction tube, how to improve the specificity of primers to avoid non-specific amplification, how to reduce the dimer formed between multiple primers, how to Regulating the concentration of PCR reaction reagents and its reaction conditions to achieve effective amplification of each target fragment limits the application of multiplex PCR technology.

Method used

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  • Multiple PCR detection kit for virus hepatitis pathogens and preparation and application thereof
  • Multiple PCR detection kit for virus hepatitis pathogens and preparation and application thereof
  • Multiple PCR detection kit for virus hepatitis pathogens and preparation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0071] Embodiment 1 is directed at the preparation of the multiple PCR detection kit of viral hepatitis pathogen:

[0072] 1. Preparation of virus positive control substance:

[0073] 1) Isolate the viral nucleic acid of hepatitis A virus, hepatitis B virus, hepatitis C virus, human cytomegalovirus, herpes simplex virus type 1, and herpes simplex virus type 6;

[0074] 2) Form a PCR reaction system, which includes multiple primers, dNTP, Mg 2+ , reverse transcription-PCR reaction solution, reverse transcriptase and DNA polymerase one-step enzyme mixture, RNase inhibitor and DEPC·H 2 O; Wherein, described multiple primer sequence and product size are shown in Table 1;

[0075] Table 1 The sequences of seven pairs of specific primers and the size of amplified products

[0076]

[0077]

[0078] 3) adding the viral nucleic acid prepared in step 1) to the mixed solution prepared in step 2) to obtain a PCR reaction solution;

[0079] 4) Carry out PCR amplification reactio...

Embodiment 2

[0102] Embodiment 2, preparation and assembly of multiplex PCR detection system:

[0103] 1. Synthesis of primers:

[0104] The primers were synthesized by ABI, an American Applied Biosystems company, and seven primer pairs (SEQ ID NO: 1-14) were specifically corresponding to hepatitis A virus, hepatitis B virus, hepatitis C virus, human cytomegalovirus, and herpes simplex virus Type 1, herpes simplex virus type 2 and herpes simplex virus type 6 viruses, as shown in Table 1. Primer pairs corresponding to each type of virus are mixed and packaged. When mixed and packaged, the mixed primers are packaged according to hepatitis A virus, hepatitis B virus, hepatitis C virus, human cytomegalovirus, herpes simplex virus type 1, and herpes simplex virus. The molar numbers of the seven primer pairs of type 2 and herpes simplex virus type 6 were mixed in a ratio of 1:1:1:1:1:1:1. At the same time, the standard virus positive control substances corresponding to various combinations pre...

Embodiment 3

[0106] Embodiment 3, the detection of positive control substance:

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Abstract

The invention belongs to the field of biological science, in particular to a multiple PCR detection kit for virus hepatitis pathogens and preparation and application thereof. The multiple PCR detection kit comprises one or combination of two to seven of seven primer pairs, and the seven primer pairs specifically correspond to hepatitis A virus, hepatitis B virus, hepatitis C virus, human cytomegalovirus, herpes simplex virus type 1, herpes simplex virus type 2 and herpes simplex virus type 6 respectively. The multiple PCR detection kit can be effectively used for detecting virus hepatitis pathogens, and has the advantages of simple and convenient production method and use and operation, high accuracy, and capacities of saving time, reagents and expenditure and providing more accurate diagnostic information for clinical service.

Description

technical field [0001] The invention belongs to the field of biological sciences, and in particular relates to a multiple PCR detection kit for viral hepatitis pathogens and its preparation and application. Background technique [0002] Viral hepatitis is a serious threat to human health, and it is also a common and frequently-occurring disease in my country. Chronic hepatitis can progress to liver fibrosis, and develop into cirrhosis, end-stage liver disease, etc. In all clinical types of hepatitis, the simple infection rate was 61.4%, the double infection rate was 32.4%, and the triple infection rate was 6.5%. It is roughly estimated that the direct medical expenses for the symptomatic treatment of hepatitis in my country exceed 100 billion yuan each year. In addition to hepatitis caused by hepatitis A, B, C, and E viruses, it has been confirmed that human cytomegalovirus (HCMV) and herpes simplex virus (HHV) can cause viral hepatitis. Most viruses that cause viral hepa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCY02A50/30
Inventor 刘晓颖邵俊杰张敬敬王迅
Owner SHANGHAI BLOOD CENT
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