RDA (Recombinase-dependent amplification) method and kit for quickly detecting Coxsachie virus 16 and enterovirus 71

A technology for coxsackie virus and enterovirus, applied in the field of molecular biology, can solve the problems such as the inability to meet the simple, rapid and accurate detection requirements of disease prevention and control, the long cycle of virus isolation and detection methods, and the high operational requirements of detection personnel. , to achieve the effect of low detection cost, high precision and rapid molecular detection, and low length requirements

Pending Publication Date: 2020-05-01
GUANGZHOU PLUSLIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the virus isolation detection method has a long cycle and is not suitable for clinical detection, and the reliability of the serum immune detection method is poor. PCR detection methods such as conventional PCR, RT-PCR, real-time fluorescent quantitative PCR, etc., play an important role in the detection of pathogenic microorganisms, but their general It requires instruments and laboratories capable of precise temperature control, and has high operating requirements for testing personnel. It is not suitable for on-site testing of samples, is not conducive to grassroots promotion, and cannot meet the simple, fast and accurate testing requirements for disease prevention and control.

Method used

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  • RDA (Recombinase-dependent amplification) method and kit for quickly detecting Coxsachie virus 16 and enterovirus 71
  • RDA (Recombinase-dependent amplification) method and kit for quickly detecting Coxsachie virus 16 and enterovirus 71
  • RDA (Recombinase-dependent amplification) method and kit for quickly detecting Coxsachie virus 16 and enterovirus 71

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 A detection kit for coxsackievirus A16 (CA16) and enterovirus 71 (EV71) RDA fluorescence method

[0093] (1) Acquisition of recombinant enzyme KX and KY proteins

[0094] The reported recombinant enzyme UvsX has poor stability and is difficult to mass produce and store for a long time. In order to solve this problem, the research and development team used bioinformatics methods to analyze and simulate large quantities of protein structures, and finally found a new recombinant enzyme Enzyme KX and its accessory protein KY.

[0095] In this example, the R&D team extracted the key functional site information in the recombinase structure, such as DNA binding site, ATP hydrolysis site, etc., and mapped it to the three-dimensional structure of the protein to obtain secondary structure information and tertiary structure information , by integrating the functional residues of the primary structure sequence, secondary structure features and tertiary structure spatial ...

Embodiment 2

[0145] Example 2 RDA fluorescence method detection reagent sensitivity test

[0146] The positive control is the pUC57 plasmid containing the conserved genes of coxsackievirus type A16 and enterovirus type 71 (EV71), and the negative control is the empty vector pUC57 plasmid.

[0147] The specific operation is as follows:

[0148] Step 1: Dilute the positive control plasmid to 10^4c, and then dilute to 10^3c, 10^2c, and 10^1c by 10-fold serial dilution.

[0149] Step two, sample processing. Take 5 μL of each concentration of the plasmid in step 1 in an EP tube, and take 5 μL of the negative control in another EP tube, add 20 μL of Buffer A respectively, shake and mix, and let stand at room temperature for 10-15 minutes;

[0150] Step 3, system preparation and testing. Add 25 μL Buffer B to each tube, shake and mix, add 50 μL of the mixed solution to the RDA fluorescence method reaction module, cover the tube cap, shake and centrifuge, and detect immediately; the reaction pr...

Embodiment 3

[0162] Example 3 RDA fluorescence method detection reagent specificity test

[0163] 1 case of Coxsackie virus 16 (Coxsachie virus 16, CA16), 1 case of enterovirus 71 (Enterovirus, EV71), 1 case of Norovirus (NV), 1 case of adenovirus (Adenovirus, AV), rotavirus (Rotavirus, RV) 5 kinds, a total of 5 cases were tested by fluorescent quantitative PCR as positive samples for the corresponding virus, and the specificity of the kit was tested.

[0164] The specific operation is as follows:

[0165] Step 1. Sample processing. Take 5 μL of each of the above 5 positive samples in EP tubes, and at the same time take 5 μL of the positive control and negative control of the kit in a new EP tube, add 20 μL Buffer A respectively, shake and mix, and let stand at room temperature for 10-15 minutes;

[0166]Step 3, system preparation and testing. Add 25 μL Buffer B to each tube, shake and mix, add 50 μL of the mixed solution to the RDA fluorescence method reaction module, cover the tube ca...

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Abstract

The invention discloses an RDA (Recombinase-dependent amplification) method and a kit for quickly detecting a Coxsachie virus 16 and an enterovirus 71. The kit comprises a specific primer pair and anRDA fluorescence labeling probe to realize a purpose of safely, specifically, sensitively and conveniently detecting the Coxsachie virus 16 (CA 16) and an enterovirus 71 (EV 71) so as to make up for deficiencies in an existing traditional detection technology. The kit provided by the method can omit a nucleic acid extraction step, and the detection of the CA 16 and the EV 71 can be realized in 20min under a constant temperature condition of 37-42 DEG C, specificity is 100%, and the kit is very suitable for field quick detection. Compared with a common PCR (Polymerase Chain Reaction) method, the RDA fluorescence method carries out reaction at a constant temperature, does not require temperature change, does not require a complex instrument and is short in reaction time. Therefore, the method and the kit thereof have the characteristics of being simple and quick in operation, good in specificity, high in sensitivity, low in cost and the like, provide an effective technical means for thefield quick detection and screening of the CA 16 and the EV 71, and have a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a pair of primers, probes and related kits for detecting Coxsackievirus A16 and Enterovirus 71 nucleic acids based on RDA fluorescence detection technology. Background technique [0002] Hand-foot-and-mouth disease (Hand-foot-and-mouth disease, HFMD) is an infectious disease caused by enteroviruses. There are more than 20 enteroviruses (types) that cause HFMD, among which Coxsackievirus A16 type ( Coxsachie virus 16, CA16) and enterovirus 71 (Enterovirus 71, EV71) are the most common. Hand, foot and mouth disease is a common infectious disease in infants and young children. It mostly occurs in children under 5 years old. heal. HFMD virus can be transmitted through direct contact with nasopharyngeal secretions, saliva, herpes fluid or feces of an infected person. Enterovirus infection, the incubation period is 2 to 10 days, with an average of 3 to 5 d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2563/107
Inventor 陈翀刘华勇谢婵芳黄嘉恩
Owner GUANGZHOU PLUSLIFE TECH CO LTD
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