Respiratory syncytial virus nucleic acid rapid detection kit based on CRISPR/Cas12a and detection method

A technology of syncytial virus and detection method, which is applied in the genetic detection of respiratory syncytial virus and the biological field, can solve the problems of being unsuitable for early diagnosis, complicated technology, difficult to judge the results, etc., and achieve high-specificity, rapid visual detection, and high sensitivity. The effect of visual detection, convenient and quick result interpretation

Active Publication Date: 2020-10-16
国家卫生健康委科学技术研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the virus isolation and culture method is known as the "gold standard", its technique is complex, time-consuming and generally takes more than a week and the cost is high, so it is not suitable for early diagnosis; immunological method is currently the most widely used technology, but this method requires Containing a certain amount of antigens and high-quality specific antibodies, infants and young children may have false negatives due to low immunity. Adult

Method used

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  • Respiratory syncytial virus nucleic acid rapid detection kit based on CRISPR/Cas12a and detection method
  • Respiratory syncytial virus nucleic acid rapid detection kit based on CRISPR/Cas12a and detection method
  • Respiratory syncytial virus nucleic acid rapid detection kit based on CRISPR/Cas12a and detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1: Rapid and sensitive detection of respiratory syncytial virus nucleic acid fragments

[0042] 1.1 Nucleic acid preparation

[0043] In this case, the RSV gene fragment refers to the F gene fragment corresponding to the A and B subtypes of RSV in the NCBI database, and the 620bp SEQ NO.1 of the F part gene of the A subtype and the F part of the B subtype were synthesized by Nanjing GenScript Company The 710bp SEQNO.2 of the gene was constructed into the pUC57 vector, named pUC57-RSVA-F and pUC57-RSVB-F, and the T7 promoter was introduced before the synthetic fragment, and the synthesized DNA fragment was transcribed into RNA by in vitro transcription reagent, named as pUC57-RSVA-F-RNA and pUC57-RSVB-F-RNA.

[0044] Utilize RT-RAA amplification primer RSVA-RT-RAA-F (SEQ NO.3) in the present invention, RSVA-RT-RAA-R (SEQNO.4), RSVB-RT-RAA-F (SEQ NO.5) and RSVB-RT-RAA-R (SEQ NO.6), referring to the RT-RAA isothermal amplification operation steps, amplified to ob...

Embodiment 2

[0063] Embodiment 2: CRISPR / Cas12a detects respiratory syncytial virus nucleic acid sensitivity

[0064] In the case of sensitivity detection, the plasmid DNA is transcribed in vitro into RNA of the RSVA / B subtype, converted to copy number according to the molecular weight, and then serially diluted 10 times to obtain 2*e7, 2*e6, 2*e5 per microliter , 2*e4, 2*e3, 2*e2, 2*e1 and 2*e0 copy number (copy / μL). 1 μL of gradient dilution samples were subjected to RT-RAA amplification reaction: 25 μL 2*Buffer, 2 μL RT-RAA-F, 2 μL RT-RAA-R and 2.5 μL magnesium acetate, mixed well, reacted at 37°C for 20 minutes, obtained samples for The next step is nucleic acid testing.

[0065] According to the results obtained in Example 1, RSVAF-crRNA-2 and RSVBF-crRNA-3 have higher sensitivity to the detection of respiratory syncytial virus genes of A and B subtypes, so these two specific crRNAs are used for subsequent detection. This test uses a 20 μL system as shown in Table 3, but is not limi...

Embodiment 3

[0071] Example 3: Rapid detection of respiratory syncytial virus nucleic acid on clinical sample nucleic acid

[0072] In this example, rapid detection of nucleic acid in clinical samples is performed. All samples and operations are completed in the laboratory. In this case, the sample is nucleic acid obtained from throat swab for CRISPR / Cas12a detection. In this example, a viral DNA / RNA extraction kit (magnetic bead method) purchased from Novizyme was used to obtain pretreated nucleic acid. The steps are as follows: wash the oral swab with PBS, add 20 μL proteinase K to the cleaning solution, add magnetic beads to incubate, let it stand at room temperature for 5 minutes, wash the magnetic beads twice with a magnetic stand, add 50 μL eluent to elute and proceed to the next step detection.

[0073] Using RT-RAA amplification primers SEQ NO.3 and SEQ NO.6 in the present invention, referring to the RT-RAA isothermal amplification operation steps, take 2 μl of each sample to be t...

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Abstract

The invention discloses a respiratory syncytial virus nucleic acid rapid detection kit based on CRISPR/Cas12a and a detection method. The CRISPR/Cas12a kit includes a CRISPR/Cas12a detection system; the CRISPR/Cas12a detection system includes a specific crRNA, CRISPR/Cas12a protein and a single-stranded DNA reporter system for respiratory syncytial virus; the specific crRNA is any one or more kinds designed and synthesized based on respiratory syncytial virus nucleic acid; and the single-stranded DNA reporter system includes a ssDNA FQ reporter for fluorescence detection of a microplate readerand/or a ssDNA DB reporter for immune colloidal gold test stripe detection. The CRISPR/Cas12a is used for detecting a nucleic acid sequence of respiratory syncytial virus for the first time, and thedetection kit has the advantages of high sensitivity, high specificity, short time-consuming, and no dependence on large-scale experimental equipment.

Description

technical field [0001] The invention relates to the field of genetic detection of respiratory syncytial virus, more specifically, to a rapid detection method and kit for respiratory syncytial virus nucleic acid based on CRISPR / Cas12a, belonging to the field of biotechnology. Background technique [0002] Acute lower respiratory tract infection is one of the leading causes of death in children worldwide, among which respiratory syncytial virus (RSV) is the most common pathogen causing acute lower respiratory tract infection every year, and it is also an important cause of nosocomial infection. RSV virus is a single-stranded RNA respiratory virus belonging to the Paramyxoviridae family. It can be transmitted by air droplets and close contact, and its infectious ability is very strong. More common in newborns and infants under 6 months. The incubation period of RSV infection is 4-5 days, and the time of shedding virus can last for 1-5 weeks. Infants and young children have se...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844G01N33/558G01N33/543G01N33/533
CPCC12Q1/701C12Q1/6844G01N33/558G01N33/54313G01N33/533C12Q2521/327C12Q2521/507C12Q2522/101C12Q2521/107
Inventor 马旭张璐王伟佳金孝华
Owner 国家卫生健康委科学技术研究所
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