CRISPR-Cas12a detection primer set and its application for tularensis

A technology of tularemia and primer set, which is applied in the biological field, can solve the problems of inability to improve the sensitivity, and achieve the effects of short detection time, good sensitivity and broad application prospects

Active Publication Date: 2020-12-29
ACADEMY OF MILITARY MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in principle, the essence of its single-stage amplification reaction has not changed. Compared with most real-time quantitative PCR (Q-PCR) detection methods currently on the market, there is no substantial improvement in sensitivity.

Method used

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  • CRISPR-Cas12a detection primer set and its application for tularensis
  • CRISPR-Cas12a detection primer set and its application for tularensis
  • CRISPR-Cas12a detection primer set and its application for tularensis

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Embodiment 1

[0052] Example 1, Design and Screening of CRISPR-Cas12a Detection Primers for Tularemia

[0053] 1. Sequence design

[0054] 1. Target sequence selection

[0055] On the basis of previous studies, the inventor selected the conserved sequence of the main membrane protein precursor (tul4) gene of Francisella tularensis as the target sequence (as shown in SEQ ID No.1) after repeated screening and comparison. As the specific conserved sequence of Francisella tularensis, the sequence can be specifically detected for Tularensis tularensis.

[0056] 2. Design of amplification primer pair and crRNA

[0057] For the specific conserved sequence of the tul4 gene of Francisella tularensis (as shown in SEQ ID No.1), many theoretically feasible RPA amplification primer pairs and crRNA were obtained by software design, but a considerable part of the effect was verified by scientific experiments Table 1 shows the sequences that are not good and some of them have good effects.

[0058] Tab...

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Abstract

The invention discloses a CRISPR-Cas12a detection primer group for francisella tularensis and an application thereof. The invention provides the primer group for detecting francisella tularensis and is composed of an RPA primer pair and crRNA; the RPA primer pair is designed according to the nucleotide sequence shown in SEQ ID NO.1 in the francisella tularensis genome; the crRNA comprises an anchoring sequence and a guide sequence, wherein the anchoring sequence can be combined with a cas protein, and the guide sequence is matched with an amplification product sequence of an RPA primer pair. The detection primer group has the characteristics of short detection time, high amplification efficiency, high sensitivity, strong specificity and the like, and the detection sensitivity on francisella tularensis positive plasmids can reach 900 copy / ml, and the detection sensitivity on the francisella tularensis genome can reach 1260 fg / mL. The francisella tularensis onsite diagnosis method has awide application prospect.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a CRISPR-Cas12a detection primer set for T. tularensis and its application, more specifically to a CRISPR-Cas12a detection primer for T. tularensis, crRNA and its application. Background technique [0002] Francisella tularensis (Francisella tularensis, or tularensis for short) is a Gram-negative bacterium that causes tularemia. Tularensis can be divided into 4 subspecies according to their biochemical properties, virulence and geographical distribution, among which the Tularensis subspecies is also called type A, and the whole arctic subspecies is called type B, which is widely distributed in the northern hemisphere and can infect More than 250 species of animals, including humans, cause outbreaks of Tularemia. Tularemia is a potential bioterror weapon and was used as a bacterial weapon during World War II and the Cold War. So far, my country's isolated strains and molecul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2521/327C12Q2531/119C12Q2525/161C12Q2563/107
Inventor 袁媛白欣茹王景林孙岩松辛文文康琳凤梓涵高姗
Owner ACADEMY OF MILITARY MEDICAL SCI
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