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Isothermal amplification method for enterovirus EV nucleic acid

A technology of enterovirus and amplification reaction, applied in the field of real-time fluorescent nucleic acid constant temperature amplification detection of enterovirus, probes and related kits, and primers, can solve the problem of real-time fluorescent nucleic acid constant temperature amplification detection of enterovirus Technical research reports and other issues

Active Publication Date: 2013-07-24
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problems faced by the application of SAT technology in the detection of different types of viruses are different, and it is necessary to specifically analyze the characteristics of the virus for special design.
At present, there is no research report on real-time fluorescent nucleic acid constant temperature amplification detection technology for enterovirus (EV)

Method used

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  • Isothermal amplification method for enterovirus EV nucleic acid
  • Isothermal amplification method for enterovirus EV nucleic acid
  • Isothermal amplification method for enterovirus EV nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0163] Embodiment 1 is used for the design of special primers and probes of real-time fluorescent nucleic acid constant temperature amplification detection enterovirus (EV)

[0164] The present invention selects no secondary structure and a highly conserved segment in the UTR gene at the 5' end of the EV virus as the amplified target sequence region (its nucleotide sequence is shown in SEQ ID NO: 1 in the sequence table), and is designed according to the primer probe Principle, using DNAStar, DNAMAN software and artificially designed special primers and probe sequences for real-time fluorescent nucleic acid constant temperature amplification detection of enterovirus (EV), the following specific sequences are obtained:

[0165] (1) a capture probe (TCO, Target Capture Oligo) that can specifically bind to the target nucleic acid (EV RNA) sequence of enterovirus (EV), the nucleotide sequence of the capture probe is: 5'TGCACACCGGATGGCCAATCCAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAA '(SEQ ID...

Embodiment 2

[0174] Embodiment 2 prepares the real-time fluorescent nucleic acid constant temperature amplification detection kit of enterovirus (EV)

[0175] Using the special primers and probes provided in Example 1, a real-time fluorescent nucleic acid constant temperature amplification detection kit for enterovirus (EV) of the present invention was obtained. The kit contains components such as capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, EV detection probe, internal standard detection probe, internal standard, M-MLV reverse transcriptase and T7 RNA polymerase .

[0176] capture probe

SEQ ID NO.: 2

T7 primer

SEQ ID NO.: 3

nT7 primer

SEQ ID NO.:4

EV detection probe

SEQ ID NO:5

internal standard detection probe

SEQ ID NO:6

internal standard sequence

SEQ ID NO:7

[0177] The capture probe exists in the nucleic acid extraction solution, the T7 primer, nT7 primer, EV detection probe, and internal...

Embodiment 3

[0210] Example 3 Real-time fluorescent nucleic acid constant temperature amplification detection of clinical sample throat swab

[0211] Use the kit of the present invention (see Example 2 for composition) to detect the enterovirus in the throat swab of the clinical sample, and the specific method comprises the following steps:

[0212] 1. Sample collection, transportation and storage

[0213] 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).

[0214] 2. Nucleic acid extraction

[0215] 2.1 Add 200 μl lysate (HEP...

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Abstract

The invention discloses an isothermal amplification method for enterovirus EV nucleic acid. The method comprises the following specific steps of: amplifying in a reaction system, wherein the reaction system contains a specific primer pair which amplifies enterovirus EV, and the primers can amplify amplifying products corresponding to characteristic sequences of enterovirus EV from a detection sample with extremely low EV copy number. The method provided by the invention can be used for amplifying the sample to be tested containing EV RNA quickly with high specificity, high sensitivity and low pollution, and the characteristics of high detection efficiency and high accuracy as well as wide application prospect.

Description

technical field [0001] The invention relates to the technical field of biological detection of viruses, in particular to primers and probes used in the real-time fluorescent nucleic acid constant temperature amplification detection of enterovirus (EV) combined with specific target capture technology and real-time fluorescent nucleic acid constant temperature amplification detection technology and related kits. 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] At present, there are more than 20 kinds of ente...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 于明朗马道亮汤嘉维居金良
Owner SHANGHAI RENDU BIOTECH
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