CRISPR-Cas12a detection primer group for francisella tularensis and application thereof

A tularemia and primer set technology, applied in the biological field, can solve problems such as inability to improve sensitivity, and achieve the effects of short detection time, high amplification efficiency and strong specificity

Active Publication Date: 2020-07-10
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This technology provides an improved way for detecting bacterial DNA sequences called Tollamicin-producing Bacilli (Tcr). It uses two types of probes - one targeted at certain parts or structures within this sequence, another designed specifically against it. These techniques are fast and efficient but also highly sensitive with respect to other organisms like Francisella tulara. They provide reliable results even when there're only small amounts of sample available from these sources. Overall, they offer great potential applications in fields such as medical diagnoses where rapid testing methods may be unavailable due to lack of sufficient samples.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving the accuracy and speed of diagnosing tuloaemia caused by certain types of microorganisms such as Tauraea spirotae. Current techniques involve isolating these organism from other sources like soil, airborne particles, etc., making them time-consuming and costly. Therefore, an improved technique needs to develop faster and more sensitive tests for identifying tulosilauximnaeus pathogenesis without requiring specialized training environments and equipment.

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  • CRISPR-Cas12a detection primer group for francisella tularensis and application thereof
  • CRISPR-Cas12a detection primer group for francisella tularensis and application thereof
  • CRISPR-Cas12a detection primer group for francisella tularensis and application thereof

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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

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Claims

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Application Information

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Owner ACADEMY OF MILITARY MEDICAL SCI
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