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Multiple real-time quantitative PCR primer, probe and detection method for identifying viral pathogens relevant to fever with eruption syndrome as infection diseases

A real-time fluorescence quantitative and symptomatic technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc., can solve the problems of untimely detection, insufficient sensitivity, time-consuming and cumbersome, etc., and achieve a high degree of automation , high sensitivity, stable results

Active Publication Date: 2011-08-03
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional isolation culture and serological detection methods are time-consuming and cumbersome, resulting in untimely detection and insufficient sensitivity. Therefore, it is urgent to develop new technologies for rapid and accurate detection to screen suspicious cases, control the spread of the epidemic, and give timely symptomatic treatment

Method used

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  • Multiple real-time quantitative PCR primer, probe and detection method for identifying viral pathogens relevant to fever with eruption syndrome as infection diseases
  • Multiple real-time quantitative PCR primer, probe and detection method for identifying viral pathogens relevant to fever with eruption syndrome as infection diseases
  • Multiple real-time quantitative PCR primer, probe and detection method for identifying viral pathogens relevant to fever with eruption syndrome as infection diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Design of primers and probes

[0048] By comparing and analyzing the gene sequences of all known viruses, select highly conserved segments, and design multiple pairs of primers and probes. The length of the primers is generally about 20 bases. The optimal primer-probe sequence combination is as follows:

[0049] Analyze the information of the above-mentioned virus-specific genes, and use the sequence analysis software DNASTAR to exclude inter-primer / inner dimers, and verify the specificity of the primers and the homology of similar pathogens by BLAST, and design primers and primers for the detection of the above-mentioned pathogens. probe.

Embodiment 2

[0050] Embodiment 2: Construction and optimization of PCR reaction system

[0051] 1. Extraction of total nucleic acid from the specimen

[0052] Each strain is provided by our laboratory. At the same time, the virus titer titration (TCID 50 / ml), TCID 50 That is, half of the infectious dose of tissue cell culture, each group of virus strains were removed 1 TCID 50 / ml of virus load.

[0053] The inactivated virus was used as the sample to be tested, and the nucleic acid was extracted using the QIAGen RNeasy Mini Kit (Qiagen, Chatsworth, CA, catalog #74104). The operation is carried out according to the instruction manual. Extraction process and main parameters:

[0054] (1) When using this kit for the first time, add 100% ethanol to the AW1 and AW2 buffers according to the volume indicated on the reagent bottle, add 25 ml absolute ethanol to 19 ml AW1, add 30 ml absolute ethanol to 13 ml AW1 Ethanol; add 28 μg / ml carrier RNA to AL.

[0055] (2) Put 25 μl Qiagen Pro...

Embodiment 3

[0077] Example 3: Sensitivity Analysis of Multiplex Fluorescent Quantitative RT-PCR

[0078] According to the above method, the virus pathogens of the above fever with rash syndrome infectious diseases (including rubella virus, measles virus, dengue virus, varicella zoster virus, human DNA parvovirus, enterovirus 71, coxsackie virus A16 ) after nucleic acid extraction, use the established multiplex real-time fluorescent quantitative RT-PCR method, and use the same reaction system and cycle parameters for detection and identification. The results showed that each group of reactions had corresponding specific fluorescence amplification curves for the nucleic acid templates of all 8 strains of viruses, and the corresponding templates of other virus strains were all negative ( Figure 1-8 ), which shows that the established multiplex real-time fluorescent quantitative PCR method has good specificity.

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Abstract

The invention discloses multiple real-time quantitative PCR primer, probe and a detection method for identifying viral pathogens relevant to fevers with eruption syndromes as infection diseases, which is used for carrying out multiple real-time fluorescent quantitative PCR detection on varicella-herpes zoster viruses, human small DNA (Deoxyribonucleic Acid) viruses B19, enteroviruses (enteroviruses 71 type and coxsackie viruses A16 type), dengue viruses, rubella viruses and measles viruses. The invention can simultaneously carry out qualitative or quantitative detection on eight kinds of human viruses in various types of samples by multiple double-tubes PCR. The detection method has the advantages of simple operation, short time consumption, high sensitivity and strong specificity, is suitable for field detection, early diagnosis, epidemics detection and research and the like, and takes the actions of assistance and identification diagnosis on the fevers with eruption syndromes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer, a probe, a kit and a detection method for detecting and identifying viral pathogens of infectious diseases of febrile rash syndrome. Background technique [0002] Fever with rash syndrome infectious disease refers to fever with rash, fever ≥ 37.5 ℃, lasting for more than one day, accompanied by systemic or local skin or mucous membrane rash. Its viral pathogens mainly include enterovirus (EV), rubella virus (Rubella virus, RV), measles virus (Measles virus, MV), dengue virus (Dengue virus, DEV), varicella zoster virus (VZV) , Human DNA Parvovirus (HPV B19), Enterovirus 71 (EV71), Coxsackievirus A16 (Cox A16), etc. [0003] Fever with rash syndrome is generally clinically diagnosed based on symptoms. Some mild and atypical cases require microbiological examination to confirm the diagnosis. Virus isolation, serological diagnosis and viral nucleic acid examination are the ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
CPCY02A50/30
Inventor 黎孟枫朱勋吴珏珩曹开源何振健袁洁李隽张定梅徐霖杨纨张赫男
Owner SUN YAT SEN UNIV
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