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Kit for detecting human enteric viruses by real-time fluorescent quantitative PCR

A technology of enteroviruses and kits, applied in the field of real-time fluorescent PCR detection kits, can solve the problems of time-consuming, laborious, and inability to handle a large number of samples at the same time, and achieve the effect of good sensitivity

Active Publication Date: 2010-02-03
广州安基因股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PCR technology overcomes the shortcomings of traditional methods that are time-consuming, laborious and unable to meet the needs of processing a large number of samples at the same time during the epidemic period. Generally, the results can be obtained within 12 hours, and it has become an important means of rapid diagnosis of enteroviruses.

Method used

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  • Kit for detecting human enteric viruses by real-time fluorescent quantitative PCR
  • Kit for detecting human enteric viruses by real-time fluorescent quantitative PCR
  • Kit for detecting human enteric viruses by real-time fluorescent quantitative PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the development of human enterovirus fluorescent PCR detection reagent

[0033] 1. Design of primers and probes: Through sequence comparison and analysis of the existing enterovirus nucleic acid sequences in the Genbank database and the nucleic acid sequences reported in domestic and foreign published literature, the conserved genes in the 5' non-coding region of enteroviruses The fragment is the amplified target site, select a highly conserved segment with no secondary structure, and use software and manual design of multiple pairs of primers and probes according to the basic principles of primer probe design.

[0034] 2. Selection of samples: According to relevant domestic and foreign literature reports, samples such as feces, serum, throat swabs, cerebrospinal fluid, and herpes fluid can be selected.

[0035] 3. Establishment and optimization of the reaction system

[0036] Sample preparation: 8 enteroviruses identified as positive by virus culture and...

Embodiment 2

[0046] Embodiment 2: human enterovirus fluorescent PCR detection kit and its use

[0047] 1. Prepare a kit including the following components: 1 tube of RNA extraction solution (50ml / tube), 24 tubes of PCR amplification reaction solution (20μl / tube), 1 tube of negative quality control (100μl / tube), 1 tube of positive quality control 1 tube of sample (100 μl / tube), 4 tubes of quantitative reference product (50 μl / tube).

[0048] 2. Specimen collection, transportation and storage

[0049] (1) Specimen collection: The object of specimen collection is clinically diagnosed cases of HFMD at the time of the outbreak and children under 5 years old in communities (villages) near the community (village) without cases or nursery institutions where the cases occurred (as healthy control population).

[0050] Specimens should be shipped frozen with necessary information, such as specimen number, date of onset, and date of specimen collection.

[0051] (2) Specimen storage and delivery: ...

Embodiment 3

[0074] Embodiment 3: Reproducibility experiment of enterovirus fluorescent PCR detection kit

[0075] at 5.0×10 4 The positive reference product of PFU / ml is as the sample to be tested, detects 5.0 * 10 simultaneously with 7 enterovirus fluorescent PCR detection kits of 3 production batches according to the method in embodiment 2 4 Positive reference substance of PFU / ml, the results are attached Figure 5 As shown: the Ct values ​​of different amplification curves are all between 27 and 28, and the calculated coefficient of variation CV=5%, indicating that the kit has good repeatability.

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Abstract

The invention relates to a real-time fluorescent PCR detection kit, in particular to a kit for diagnosing human enteric virus infection early and quickly by real-time fluorescent quantitative polymerase chain reaction technique. The kit has quite high sensitivity and specificity, and can realize quick and early diagnosis and quantitative analysis of human enteric viruses in samples of feces, serum, throat swab, cerebrospinal fluid, herpes fluid and the like.

Description

technical field [0001] The invention relates to a real-time fluorescent PCR detection kit, in particular to a kit for early and rapid diagnosis of human enterovirus infection by real-time fluorescent quantitative polymerase chain reaction technology. The detection and quantitative analysis of human enterovirus in feces, serum, throat swab, cerebrospinal fluid, herpes fluid and other samples can be realized by the kit of the invention. Background technique [0002] Enteroviruses (EV) belong to the family Picornaviridae, which have the same physical, chemical and biological characteristics, including poliovirus (Poliomyelitis Virus, PV), Coxsackie virus (CV) ), enterocytopathic human orphan virus (enterocytopathic human orphan virus, referred to as Echo virus, ECHOV) and more than 70 serotypes such as new enteroviruses, this genus of virus infection is widely distributed all over the world, and there are more epidemics in summer and autumn , the virus types that are prevalent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCY02A50/30
Inventor 邓中平高秀洁王秋泉李杰胡守旺李明程钢何蕴韶
Owner 广州安基因股份有限公司
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