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Herpes virus fluorescent PCR detection kit

A detection kit and herpes virus technology, which are applied in the direction of determination/inspection of microorganisms, microorganisms, biochemical equipment and methods, etc., can solve the problem of low sensitivity, etc., and achieve the effect of simple and convenient operation, low cost, and saving consumables

Inactive Publication Date: 2013-06-05
GUANGZHOU GIR MEDICINE CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serological examination uses the corresponding antibody and antigen to detect pathogens or pathogen-related proteins. This method is fast, simple and easy to operate, but the sensitivity is not high

Method used

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  • Herpes virus fluorescent PCR detection kit
  • Herpes virus fluorescent PCR detection kit
  • Herpes virus fluorescent PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: the specific experiment of primer, probe

[0050] After comparative analysis of the herpesvirus sequence data in the existing database using bioinformatics and biological software for designing primers and probes, the primers and probes shown in Table 1 were designed to achieve single or multiplex fluorescent PCR of herpesviruses. detection. Table 1 is the herpes virus EBV, HHV-6, HHV-7 primer and probe sequences.

[0051] Table 1

[0052]

[0053] In order to verify the specificity of the designed primers and probes, a single-plex fluorescent PCR reaction was designed first, and the primers and probes were evaluated, that is, the specificity of the three sets of primers and probes for EBV, HHV-6 and HHV-7 were detected separately. sex. The experiment was carried out with positive clinical samples of EBV, HHV-6, and HHV-7 viruses. The positive samples came from the National Key Experiment of Respiratory Diseases of Guangzhou Institute of Respiratory...

Embodiment 2

[0058] Embodiment 2: Specificity experiment of herpes virus multiple detection reagent

[0059] The multiple detection reagents are prepared using the PCR buffer in TAKARA'Premix Ex Taq (Perfect Real Time)', and the primers and three probes of EBV, HHV-6 and HHV-7 in Table 1 are prepared as a tube of reaction solution , the volume of the prepared reagent is 18 μl / reaction, the amount of each primer used is in the range of 7-15 pmol, and the amount of each probe used is 0.5-5 pmol; 2 μl is reserved for adding the enzyme mixture, and 5 μl is reserved for adding the template; The total volume was 25 μl / reaction.

[0060] Single positive template (EBV, HHV-6 or HHV-7); double positive template (mixed EBV and HHV-6; mixed EBV and HHV-7 or mixed HHV-6 and HHV-7); triple positive template (mixed EBV and HHV-7) , HHV-6 and HHV-7 mixed) to perform experiments on multiplex detection reagents, while using single-plex fluorescent PCR reagents for comparison. The experimental results a...

Embodiment 3

[0064] Embodiment 3: Sensitivity experiment of herpes virus multiple detection reagent

[0065] In order to further test the sensitivity of the reagents, the PCR positive amplified fragments of EBV, HHV-6 and HHV-7 were cloned into pMD-18T vector (TAKARA), and transformed, cultured, screened, and extracted as positive product verification reagents sensitivity. The positive product was calculated by detecting its absorbance value, and the number of molecules was calculated and diluted 10 times, which was a dilution of 1 to 6, respectively indicating that the EBV, HHV-6 and HHV-7 viruses were diluted 10 to 10 times. 6 times (10 6 ~10 copies / mL), compared with multiple herpes virus detection reagents and single detection, the results of quantitative PCR experiments are shown in Table 4. Experiments have proved that the sensitivity of the three reagents can reach 100 copies / mL, and the sensitivity of the triple detection reagent is basically the same as that of the single det...

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Abstract

The invention provides a herpes virus fluorescent PCR detection kit which contains a primer with a sequence selected from SEQ ID NO: 1-6, and a probe with a sequence selected from SEQ ID NO: 7-9. The herpes virus detection kit of the invention comprises a plurality of kits, such as an EBV, HHV-6, HHV-7 single-detection kit, an EBV, HHV-6 combined-detection kit, an EBV, HHV-7 combined-detection kit, and an EBV, HHV-6, HHV-7 combined-detection kit. The invention adopts the specific primer and the Taqman probe, realizes single detection or multiple detection of herpes virus, has the advantages of strong specificity, high sensitivity, high speed, simple and convenient operation, consumable saving, low cost, and the like, can be used as a kit for detecting EBV, HHV-6, and HHV-7, and can be used for scientific research and clinical application.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a herpes virus fluorescent PCR detection kit, including human herpes virus type 4, also known as Epstein-Barr virus (Epstein-Barr virus, EBV), human herpes virus type 6 (Human herpesvirus 6, HHV-6), human herpesvirus 7 (Human herpesvirus 7, HHV-7) fluorescent PCR single detection and joint detection kits. [0002] Background technique [0003] Herpesviruses cause many clinically important human diseases. Among the herpes viruses, Epstein-Barr virus belongs to the Gammaherpes virus family. After the body is infected with Epstein-Barr virus, it can produce infectious mononucleosis, Epstein-Barr virus-associated hemophagocytic syndrome and X-linked lymphoproliferative disease and other diseases. It is widely infected in the population. According to serological surveys, the positive rate of EB virus VCA-IgG antibody in children aged 3 to 5 in my country is over...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 周荣白培胜刘文宽梁焕喜高文娟
Owner GUANGZHOU GIR MEDICINE CO LTD
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