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Coxsackie virus type A16 (CA16) real-time fluorescent nucleic acid isothermal amplification detection kit

A Coxsackie virus, CA16 technology, applied in DNA/RNA fragmentation, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of amplicon contamination, low sensitivity, long detection cycle, etc.

Active Publication Date: 2013-11-13
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems that the existing Coxsackievirus A16 type (CA16) detection method has low sensitivity, long detection cycle, easy to cause contamination of amplified products, false positive or false negative of experimental results and high detection cost, the present invention Provides a Coxsackievirus A16 (CA16) real-time fluorescent nucleic acid constant temperature amplification detection technology with short detection cycle, high sensitivity, high specificity, low pollution, stable reaction, low detection cost, and easy popularization and application, including special primers , probes, kits and their use

Method used

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  • Coxsackie virus type A16 (CA16) real-time fluorescent nucleic acid isothermal amplification detection kit
  • Coxsackie virus type A16 (CA16) real-time fluorescent nucleic acid isothermal amplification detection kit
  • Coxsackie virus type A16 (CA16) real-time fluorescent nucleic acid isothermal amplification detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Embodiment 1, for real-time fluorescent nucleic acid constant temperature amplification detection Coxsackie virus A16 type (CA16) special primer and probe design

[0099] The present invention selects no secondary structure and a highly conserved segment in the CA16 virus VP1 gene as the amplified target sequence region (its nucleotide sequence is shown in sequence 1 in the sequence table), and according to the principle of primer probe design, DNAStar, DNAMAN Software and manual design are used for real-time fluorescent nucleic acid constant temperature amplification to detect the special primer and probe sequence of Coxsackie virus A16 type (CA16), and obtain the following specific sequence:

[0100] (1) A capture probe (TCO, Target Capture Oligo) that can specifically bind to the target nucleic acid (CA16 RNA) sequence of Coxsackievirus A16 type (CA16), the nucleotide sequence of the capture probe is: 5 'AGTTTGCTCAATGTCCTCTGTGTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA3' (seque...

Embodiment 2

[0104] Embodiment 2, prepare the real-time fluorescent nucleic acid constant temperature amplification detection kit of Coxsackie virus A16 type (CA16)

[0105] Using the special primers and probes provided in Example 1, a real-time fluorescent nucleic acid constant temperature amplification detection kit for Coxsackievirus type A16 (CA16) of the present invention was obtained. The kit contains components such as capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, CA16 detection probe, internal standard detection probe, internal standard, M-MLV reverse transcriptase and T7 RNA polymerase .

[0106] The capture probe exists in the nucleic acid extraction solution, the T7 primer, nT7 primer, CA16 detection probe, and internal standard detection probe exist in the CA16 detection solution, and the M-MLV reverse transcriptase and T7 RNA polymerase The enzyme exists in the SAT enzyme solution. Specifically, the kit is divided into A box (specimen processing unit) stor...

Embodiment 3

[0139] Example 3. Real-time fluorescent nucleic acid constant temperature amplification detection of clinical sample throat swab

[0140] The Coxsackie virus A16 type (CA16) in the clinical sample throat swab is detected with the kit of the present invention (see Example 2 for composition), and the specific method comprises the following steps:

[0141] 1. Sample collection, transportation and storage

[0142] The clinician will collect the specimens according to the actual situation. The test specimens are throat swabs. The collection method is: wipe the posterior pharyngeal wall and the pharyngeal tonsils on both sides with a special sampling cotton swab, invade the swab into 3-5mL normal saline, and send it sealed for inspection. Within 48 hours after the sample is collected, store it at 4°C and send it to the enterovirus monitoring network laboratory (or store it at -70°C for testing, and deliver it within one week).

[0143] 2. Nucleic acid extraction

[0144] 2.1 Add 2...

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Abstract

The invention discloses a Coxsackie virus type A16 (CA16) real-time fluorescent nucleic acid isothermal amplification detection kit comprising reagents such as a capturing probe, CA16 amplification primers T7 primer and nT7 primer, a CA16 detection probe, M-MLV reverse transcriptase, T7 RNA polymerase, and the like. The kit can be used for detecting CA16 RNA in throat swab or stool, and has the characteristics of high specificity, high sensitivity (reaching 10copies / reaction), low pollution (amplification product RNA can be easily degraded under natural environment), and fast detection (conventionally detection can be finished within 60min). The kit can perform important effect in clinical diagnosis of CA16 early-stage infection, and can be widely applied.

Description

technical field [0001] The invention relates to the technical field of biological detection of viruses, in particular to a kit for detecting Coxsackievirus A16 (CA16) by using magnetic bead-RNA enrichment technology to extract and purify target RNA and real-time fluorescent nucleic acid constant temperature amplification detection technology . The detection of CA16 in samples such as throat swabs and feces can be realized by the kit of the present invention. Background technique [0002] Hand, foot and mouth disease is a global infectious disease that mostly occurs in infants and young children. It can cause herpes on the hands, feet, and mouth. In a small number of patients, it can cause fatal complications such as myocarditis, pulmonary edema, and meningoencephalitis. In my country, hand, foot and mouth disease was first discovered in Shanghai in 1981; since then, it has been reported all over the country. In some areas the prevalence is as high as 1000 / 100,000. [0003...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 于明辉汤嘉维居金良
Owner SHANGHAI RENDU BIOTECH
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