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Universal primer, probe and test kit for intestinal virus nucleic acid testing

A technology of enteroviruses and universal primers, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as difficult probe design, achieve high detection rate, high sensitivity, The effect of strong probe degeneracy

Pending Publication Date: 2020-08-14
江苏派森杰生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mutation of the virus brings difficulties to the design of primers and probes. In order to accurately identify the circulating enteroviruses, it is necessary to regularly compare and analyze the sequences of the circulating strains

Method used

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  • Universal primer, probe and test kit for intestinal virus nucleic acid testing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: Design and synthesis of primers and probes

[0018] One of the keys of the present invention is to design a set of primers and probes that can be used for general nucleic acid detection of enteroviruses. According to the genome sequences of enteroviruses in GenBank database, the present invention uses MAFFT software to compare and analyze the consistency of gene sequences of different types of enteroviruses, and select relatively conserved regions to design whole-genome specific amplification primer sequences. The designed primers have matching sequences for most enteroviruses. Based on the fact that there are more than 100 types of enteroviruses, and new enteroviruses such as D68, C105, and C107 have emerged in recent years, degenerate primers have been introduced. . Due to the variation site of the enterovirus D68 serotype, the present invention introduces a "Y" degenerate primer at the third base of the forward primer (EV-UN-R). Due to the mutation of...

Embodiment 2

[0023] Embodiment 2: the extraction of RNA

[0024] Enterovirus samples were taken as enterovirus cell culture, herpes fluid, stool samples, anal swabs and throat swabs. Use the High Pure Viral RNA Kit (High Pure Viral RNA Kit) to extract the RNA in the enterovirus samples, and the specific extraction steps are carried out according to the operating instructions. Among them, enterovirus cell culture and RNA extraction in herpes fluid are directly extracted according to the instructions. Fecal samples, anal swabs, and throat swab samples need to be pretreated. Add 0.01M PBS in advance, pH7.5; Add 5mL PBS to feces samples (0.5ml liquid feces), then centrifuge the suspension at 12000×g for 10min, take the supernatant for RNA extraction, elute with 50μl DEPC water, and put in - Store at 80°C.

Embodiment 3

[0025] Embodiment 3: the establishment of Real-time RT-PCR amplification method

[0026] Using the enterovirus RNA extracted in Example 2 as a template, a real-time RT-PCR reaction was performed (reagents were purchased from TaKaRa Company).

[0027] The fluorescent quantitative RT-PCR reaction system is as follows:

[0028] The 25 μl rRT-PCR detection system contains: 2×OneStep RT-PCR Buffer 12.5 μl, 10 μM forward primer (SEQ ID No: 1) 3.0 μl, 10 μM reverse primer (SEQ ID No: 2) 3.0 μl, 10 μM probe Needle (SEQ ID No:3) 0.5μl, Rox Reference Dye Ⅱ (50×) 0.5μl, 5U / μl Ex Taq HS 0.5μl, 40U / μl PrimeScriptRT enzyme Mix 0.5μl, sample RNA to be detected 4.5μl. Put the reaction tube of the above reaction system into a fluorescent quantitative PCR instrument, and set the rRT-PCR reaction conditions as follows: reverse transcription at 50°C for 20 minutes, reaction at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing / extension at 60°C for 40 seconds, and PCR amplificati...

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Abstract

The invention relates to the technical field of biological detection, in particular to a universal primer, a probe and a test kit for intestinal virus nucleic acid testing. The primer and the fluorescent probe are designed according to a new mutation site of an intestinal virus 5'UTR sequence, the primer and the probe for the universal nucleic acid testing of the intestinal virus provided by the invention are strong in degeneracy, and a degeneracy base is introduced at the 8-base position of the 3' end of an upstream prime; and a degenerate base is introduced at the 16-base position of the 5'end of a downstream primer according to T base mutation. RRT-PCR detection is carried out by adopting the primer and the probe disclosed by the invention, so that the primer and the probe have relatively high detection rate and high sensitivity on the novel intestinal virus generated by mutation of the intestinal virus 5'UTR sequence; and a scientific basis is provided for diagnosis of related diseases of intestinal virus infection.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a universal primer, a probe and a kit for the detection of enterovirus nucleic acid. [0002] technical background [0003] Enteroviruses belong to the small RNA virus family, and there are four species within the genus Enteroviruses that can infect humans: A, B, C, and D, respectively. Enteroviruses are further divided into multiple serotypes based on genetic and phenotypic similarities, and there are more than 110 serotypes so far. Enteroviruses are prone to errors during genome replication and mutate over time. The recombination phenomenon between enteroviruses is very common, and the recombination further increases the genetic diversity, which also brings difficulties to clinical diagnosis. [0004] The identification of enterovirus types by Real-time RT-PCR (rRT-PCR) method can confirm the diagnosis of suspected infection samples in a short period of time. When...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11
CPCC12Q1/701C12Q1/6858C12Q2600/166C12Q2521/107C12Q2531/113C12Q2563/107C12Q2545/113Y02A50/30
Inventor 申红星刘庭君王华
Owner 江苏派森杰生物科技有限公司
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