Kit for detecting respiratory syncytial virus and application thereof

A syncytial virus and kit technology, which is applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problem that the detection sensitivity of RPA technology has not been effectively improved, and the detection operation of enzyme-linked immunosorbent assay Complex, high detection costs, to achieve good application prospects, short detection time, easy to operate.

Pending Publication Date: 2022-07-29
GUANGZHOU DEAOU MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzyme-linked immunosorbent assay detection operation is complicated, the detection time is long, the probability of interaction reaction is high, and the detection cost is high
Compared with enzyme-linked immunosorbent assay, real-time fluorescence quantitative PCR has many advantages such as simple operation, high detection specificity, and high sensitivity. Expensive instruments are assisted with detection, which can only be performed in professional equipment and instruments, and high temperature PCR amplification reaction is required to achieve the detection purpose
[0006] However, there are still many problems in the direct use of RPA technology for detection, especially the problem of false positives that needs to be solved urgently. In addition, the detection sensitivity of RPA technology has not been effectively improved compared with PCR technology.

Method used

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  • Kit for detecting respiratory syncytial virus and application thereof
  • Kit for detecting respiratory syncytial virus and application thereof
  • Kit for detecting respiratory syncytial virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] In this example, the amplification efficiency of the RPA amplification primer pair for respiratory syncytial virus was studied. Under the condition that the contents of each component of the reaction system did not change, the amplification efficiency of three pairs of respiratory syncytial virus RPA primer pairs was studied.

[0048] The amplification reaction system was: 1 μL RSV-F (10 μM), 1 μL RSV-R (10 μM), 0.3 μL 1×syto-9, 20.5 μL RPA Buffer, 1.2 μL Mg(CH3COO)2 (280 mM), template (containing respiratory tract cytovirus detection target sequence T vector) 50ng, make up to 25μL with ultrapure water. The amplification program was set to 42°C, pre-denaturation for 30s; denaturation at 42°C for 15s, annealing and extension at 37°C for 15s, with 40 cycles.

[0049] The sequences of RPA amplification primer pairs for respiratory syncytial virus are shown in Table 1.

[0050] Table 1 Respiratory syncytial virus RPA amplification primer pair sequences

[0051]

[005...

Embodiment 2

[0054] In this example, 50 clinically negative samples of respiratory syncytial virus were used as specificity evaluation indicators to analyze the specificity of the RPA amplification primer pair for respiratory syncytial virus provided in Example 1. According to the RPA amplification reaction system and amplification procedure described in Example 1, 50 clinically negative samples of respiratory syncytial virus were amplified, and a positive control and a negative control were set at the same time. Among them, the template of the positive control is a T vector containing the target sequence of respiratory syncytial virus detection; the template is not added to the negative control.

[0055] Test results such as image 3 shown, according to image 3 , only the positive control showed an "S"-shaped amplification curve, and the negative control and 50 clinically negative samples did not show amplification. It shows that the RPA amplification primer pair provided by the presen...

Embodiment 3

[0057] This embodiment provides a kit for detecting respiratory syncytial virus, including an RPA reaction system and a CRISPR-Cas13a protease detection system.

[0058] The RPA reaction system is shown in Table 2.

[0059] Table 2 RPA reaction system

[0060]

[0061]

[0062] The CRISPR-Cas13a protease detection system is shown in Table 4.

[0063] Table 3 Base sequences required for CRISPR-Cas13a protease detection

[0064]

[0065] Table 4 CRISPR-Cas13a protease detection system

[0066] Reagent ingredients Amount used (μL) NTP Buffer Mix 10 T7 transcriptase 3 sgRNA 1 report sequence 5 Mg(CH 3 COO) 2

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Abstract

The invention relates to a kit for detecting a respiratory syncytial virus and application of the kit. The kit comprises an RPA amplification reaction system and a CRISPR-Cas13a protease detection system, wherein the RPA amplification reaction system comprises an RPA amplification primer pair, and the CRISPR-Cas13a protease detection system comprises sgRNA (small guide ribonucleic acid), Cas13a protein and a probe. The invention further provides a corresponding detection method, the respiratory syncytial virus can be detected through the kit under the constant-temperature condition, and the kit has the advantages of being short in detection time, easy and convenient to operate, mild in detection condition, free of assistance of expensive professional instruments and the like. The kit and the corresponding detection method have good application prospects and have the potential to be applied to clinical detection.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a kit for detecting respiratory syncytial virus and its application. Background technique [0002] Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus of the family Paramyxoviridae. Respiratory syncytial virus is a single-stranded RNA virus with two types, A and B. It is easily inactivated in an acid-base environment and can survive for several hours in a daily environment. It is often spread through droplets or close contact. It is more common in the north in the cold winter and spring, and the peak of the epidemic in the south is mainly in the hot summer and autumn. The main symptoms are fever, cough, wheezing, and X-ray examination is a small patchy shadow. Children with mild disease generally have a disease course of 6-10 days. Infants and young children under 6 months of age are mostly severe cases after being infected with respiratory syncytial...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12N15/113C12R1/93
CPCC12Q1/701C12Q1/6844C12N15/1131C12N2310/20C12Q2521/507C12Q2525/151C12Q2525/161C12Q2563/107C12Q2521/327C12Q2527/127
Inventor 陈皇佑任文玉
Owner GUANGZHOU DEAOU MEDICAL TECH CO LTD
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