Real-time fluorescence quantitative PCR detection kit for human enterovirus 71

An enterovirus and kit technology, which is applied in the field of real-time fluorescent PCR detection kits, can solve the problems of time-consuming, laborious, inability to process a large number of samples at the same time, and achieve the effect of good sensitivity

Active Publication Date: 2009-11-18
DAAN GENE CO LTD
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

RT-PCR technology overcomes the shortcomings of traditional methods that are time-consuming, laborious and unable to meet the needs of pro

Method used

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  • Real-time fluorescence quantitative PCR detection kit for human enterovirus 71
  • Real-time fluorescence quantitative PCR detection kit for human enterovirus 71
  • Real-time fluorescence quantitative PCR detection kit for human enterovirus 71

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: the development of human enterovirus 71 fluorescent PCR detection reagent

[0034] 1. Design of primers and probes: through sequence comparison and analysis of the existing EV71 nucleic acid sequences in the Genbank database and the nucleic acid sequences reported in published literature at home and abroad, the VP1 gene fragment of the EV71 virus was used as the amplification target site, Select a segment with no secondary structure and a high degree of conservation, and design multiple pairs of primers and probes using software and manually according to the basic principles of primer-probe design.

[0035] 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.

[0036] 3. Establishment and optimization of the reaction system

[0037] Sample preparation: EV71 virus identified as positive by virus culture and sequencing is use...

Embodiment 2

[0047] Embodiment 2: human enterovirus 71 fluorescent PCR detection kit and its use

[0048] 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).

[0049] 2. Specimen collection, transportation and storage

[0050] (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).

[0051] Specimens for virus isolation include feces, throat swabs, herpes fluid, and cerebrospinal fluid if the patient has neurological symptoms. Clinical specimens should avoid repeat...

Embodiment 3

[0075] Example 3: Quantitative detection of clinical samples using the EV71 virus fluorescent PCR detection kit

[0076] The clinical samples were from the Guangdong Provincial Center for Disease Control and Prevention, including feces, throat swabs, serum, cerebrospinal fluid and other sample types; the RNA extraction, PCR reaction and result analysis of the samples were carried out with reference to Example 2.

[0077] After the PCR reaction, adjust the analysis parameters according to the amplification curve to make the standard curve under the standard curve (Std curve) window reach the best (ie, the absolute value of the correlation value > 0.97), and then analyze the clinical samples. The test results of clinical samples are attached Figure 5 Shown: The Ct values ​​of the amplification curves of the 5 clinically positive specimens are 16.73, 19.78, 23.50, 24.02, and 25.93, respectively. Combined with the obvious exponential growth period of the amplification curves, all...

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Abstract

The invention relates to a real-time fluorescence quantitative PCR detection kit for human enterovirus 71, in particular to a kit for rapidly diagnosing the human enterovirus 71 infection at the early stage by using the real-time fluorescence quantitative polymerase chain reaction technology. The detection and the quantitative analysis to the human enterovirus 71 in various samples of feces, blood serum, throat swab, cerebrospinal fluid, herpes fluid, and the like can be realized through the kit.

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 71 infection by using real-time fluorescent quantitative polymerase chain reaction technology. The detection and quantitative analysis of human enterovirus type 71 in feces, serum, throat swab, cerebrospinal fluid, herpes fluid and other samples can be realized by the kit of the invention. Background technique [0002] Enterovirus 71 (EV71) is a member of the genus Enterovirus in the family Picornaviridae, and its infection mainly causes hand-foot-and-mouth disease (HFMD) and herpes Angina. Usually, HFMD caused by EV71 infection is indistinguishable from HFMD caused by Coxsackie virus A16 (CoxsackieA16, CA16) in terms of clinical symptoms, but EV71 infection can also cause aseptic meningitis ( Aseptic meningitis), brainstem encephalitis (brainstem encephalitis) and poliomyelitis-like paralysis (poliomyelitis-li...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 邓中平王秋泉高秀洁李杰胡守旺李明程钢何蕴韶
Owner DAAN GENE CO LTD
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