A kit, detection method and application for detecting nucleic acid of respiratory pathogens

A respiratory and kit technology, applied in the biological field, can solve the problems of inconvenient detection and dependence on large-scale instruments, and achieve the effects of convenient detection, high sensitivity, specificity and sensitivity.

Active Publication Date: 2021-05-14
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The technical problem to be solved by the present invention is to overcome the defects such as the existing methods for detecting respiratory pathogens (such as novel coronavirus (SARS-CoV-2), influenza A, influenza B) relying on large-scale instruments, inconvenient detection, etc., providing A kit for detecting nucleic acids of respiratory pathogens, a detection method for nucleic acids of respiratory pathogens, an isolated nucleic acid, crRNA, primer pairs and applications thereof

Method used

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  • A kit, detection method and application for detecting nucleic acid of respiratory pathogens
  • A kit, detection method and application for detecting nucleic acid of respiratory pathogens
  • A kit, detection method and application for detecting nucleic acid of respiratory pathogens

Examples

Experimental program
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Effect test

Embodiment 1

[0112] 1. Expression and purification of Cas12a protein

[0113] The Cas12a (LbCas12a, amino acid sequence shown in SEQID NO:57) gene of Lachnospiraceae bacterium ND2006 origin passes through after codon optimization (optimized sequence is shown in SEQ ID NO:102), connects into pET28a plasmid (Thermo Fisher Scientific, Massachusetts , USA), protein expression was regulated by the T7 promoter. The C-terminus of LbCas12a contains a His6 tag for affinity purification, and a TEV restriction site is inserted between the Cas12a and His6 sequences. The resulting recombinant plasmid (hereinafter referred to as pET28a-LbCas12a) was transformed into Escherichia coli BL21 (DE3) cells. The next day, the monoclonal cells were inoculated into Luria-Bertani (LB) medium containing 50 μg / mL kanamycin, and cultured with shaking at 37°C. in OD 600 When it reached 0.8, 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to induce protein expression, and cultured at 37°C for 16 hours. ...

Embodiment 2

[0153] The results obtained when testing different types of samples are as follows: Figure 20 Shown, detection method is the same as embodiment 1. It can be seen from the figure that the crRNA and primer pairs in Example 1 can be detected for different sample types (including sputum supernatant, nasal swab, throat swab, sputum, anal swab, feces, etc.), and its detection high specificity and sensitivity.

Embodiment 3

[0154] Example 3 For the detection of each pathogen in samples derived from living tissue and containing multiple pathogens

[0155] Adenovirus serotype 5 (Ad5) encoding ACE2 was transfected into BALB / c mice by intranasal (IN); 5 days later, the new coronavirus was transfected into mice by intranasal, 1 To 7 days, samples were collected for virus titer and nucleic acid detection (the detection method was the same as in Example 1). The result is as Figure 21 As shown, it can be seen from the figure that in addition to detecting the new coronavirus (N and S genes), adenovirus can also be detected. It can be seen that the crRNA and primer pair in Example 1 can detect each pathogen in a sample containing multiple pathogens derived from living tissue.

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Abstract

The present invention provides a kit for detecting nucleic acids of respiratory pathogens, which includes a CRISPR-Cas detection system: crRNA, Cas protein and nucleic acid probes as described in the present invention. The invention also provides a nucleic acid detection method for respiratory pathogens, an isolated nucleic acid, crRNA, primer pairs and applications thereof. The test kit and detection method of the present invention do not rely on large-scale instruments and can directly observe the results by naked eyes, and the detection can be realized under mild conditions, and the detection is more convenient; the use of the test kit and detection method of the present invention can be used for efficient and rapid Detect / diagnose respiratory pathogens (such as influenza A, influenza B and new coronavirus, etc.), with high specificity and sensitivity, and can be used for detection and screening of respiratory pathogens, such as rapid distinction including influenza A and influenza B And a variety of respiratory pathogens including the new coronavirus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit, a detection method and an application for detecting nucleic acid of a respiratory pathogen. Background technique [0002] Cases of COVID-19 quickly appeared in countries around the world, and was declared a worldwide public health threat by the World Health Organization (WHO). Although the median onset time of COVID-19 is currently reported to be 4-5 days, a small proportion of infected patients develop symptoms after 14 days of isolation. The most common symptoms of COVID-19 are cough and fever. However, a certain proportion of patients do not show symptoms, including X-ray detection and CT detection. The uncertainty of the time of onset of symptoms of COVID-19 and the situation of asymptomatic infection have brought great obstacles to the early and accurate detection of the disease, thus making disease prevention and control extremely challenging. This makes t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12N15/113
CPCC12Q1/6844C12Q1/701C12Q2600/16C12Q2531/119C12Q2521/107C12Q2521/301C12Q2537/143Y02A50/30
Inventor 刘佳
Owner SHANGHAI TECH UNIV
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