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CRISPR detection primer group for bordetella pertussis and application of CRISPR detection primer group

A technology for detecting primers and pertussis, which is applied in the direction of microorganism-based methods, microorganism measurement/inspection, microorganisms, etc., can solve problems such as inability to improve sensitivity, and achieve the goal of getting rid of the dependence on complex temperature-variable amplification instruments, simplifying experimental operations, and improving sensitivity. high effect

Active Publication Date: 2020-02-14
广州微远医疗器械有限公司 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 (qPCR) detection methods currently on the market, there is no substantial improvement in sensitivity.

Method used

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  • CRISPR detection primer group for bordetella pertussis and application of CRISPR detection primer group
  • CRISPR detection primer group for bordetella pertussis and application of CRISPR detection primer group
  • CRISPR detection primer group for bordetella pertussis and application of CRISPR detection primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Design of primer sequences for CRISPR detection of Bordetella pertussis.

[0042] 1. Target sequence selection.

[0043] With the development of molecular biology, some scholars have used PCR and other methods to conduct gene detection research on Bordetella pertussis. Most of these studies focus on pertussis gene insertion sequences IS481, IS1002, pertussis toxin (PT) and target gene BD485, etc. fragment. Among them, the insertion sequence IS481 has 238 copies in the genome, accounting for 6.2% of the total genome size, and this feature can greatly improve the detection sensitivity. However, studies have found that the IS481 insertion sequence also exists in the genome of Bordetella hallii, which causes pertussis-like syndrome, but usually coexists with B. pertussis infection. The inventor found through a large amount of literature research and practical statistics that no single case of Bordetella hallii has been reported, and the symptoms of clinically infected wit...

Embodiment 2

[0052] 1. The amplification efficiency screening of RPA amplification primers.

[0053] In order to screen the RPA amplification primers of Cas13a, carry and insert the bordetella pertussis IS481 sequence plasmid in the above-mentioned embodiment 1 as the standard sample to be tested, wherein the plasmid concentration is 1000copies / μl, and the screening primers include the above-mentioned primer pair 1, Primer pair 2, primer pair 3, primer pair 4, primer pair 5.

[0054] 1.1 Method.

[0055] 1) RPA amplification

[0056] RPA reaction system is 50 μL: including RPA upstream primer 0.5-2.5 μL (concentration 10 μM), RPA downstream primer 0.5-2.5 μL (concentration 10 μM), RPA enzyme premix 41.5 μL, magnesium acetate 0.5-2 μL (concentration 280 mM), to be Measure 2-5 μL of genomic DNA of the sample.

[0057] The above RPA enzyme premix contains: creatine phosphate (concentration 20-80mM), creatine kinase (concentration 50-150mM), dNTPs (concentration 100-300μM), ATP (concentrati...

Embodiment 3

[0080] In this example, sensitivity detection is performed based on RPA amplification, T7 in vitro transcription and Cas13a.

[0081] The plasmid with the conserved sequence of Bordetella pertussis IS481 was used as a template, and the calculated dilutions were 3000copies / μL, 300copies / μL, 30copies / μL, 3copies / μL, 0copy / μL, a total of 5 gradients were used as templates for sensitivity detection.

[0082] 1. Method.

[0083] 1) Two-step method.

[0084] Referring to the method in Example 2 above, after RPA amplification, T7 transcription and CRISPR reaction were performed.

[0085] 2) One-step method.

[0086] Reaction system: upstream primer (0.1-0.6μM), RPA downstream primer ((0.1-0.6μM)), RPA enzyme master mix 41.5μL, magnesium acetate (10-20mM), probe (50-400nM), NTP mixture (0.2-6mM), T7 RNA polymerase master mix (1μl, from NEB company), Cas13a protein (concentration is adjusted according to the Cas13a protein of different bacterial sources), guide RNA (crRNA, concentra...

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Abstract

The invention relates to a CRISPR detection primer group for bordetella pertussis and an application of the CRISPR detection primer group, which belong to the technical field of gene detection of CRISPR technologies. The primer group comprises an amplification primer pair and crRNA, wherein the amplification primer pair is used for amplifying a sequence of bordetella pertussis as shown in SEQ ID NO.1; wherein the crRNA comprises an anchoring sequence and a guide sequence, the anchoring sequence and the Cas protein are specifically recognized, and the guide sequence is matched with the targeting sequence fragment in the SEQ ID NO.1 sequence. By adopting the primer group, the bordetella pertussis is detected by a CRISPR technology, so that the detection time of the bordetella pertussis is shortened, and the detection can be completed within 60 minutes. According to the invention, a specific sequence combination obtained by screening is used as the primer group for detection, so that theprimer group has the advantages of high sensitivity and strong specificity, and the detection limit can reach 3 copies. The primer group is used for CRISPR detection of bordetella pertussis, dependence on qPCR instruments and other complex variable-temperature amplification instruments is avoided, and the primer group has wide application prospects.

Description

technical field [0001] The invention relates to the technical field of gene detection based on CRISPR technology, in particular to a CRISPR detection primer set for Bordetella pertussis and its application. Background technique [0002] Pertussis (pertussis / whooping cough) is an acute respiratory infection caused by Bordetella pertussis, characterized by severe and persistent cough. Whooping cough, although considered a childhood disease, can occur in all age groups. Pertussis remains an important cause of infant morbidity and mortality in developing countries; until the introduction of pertussis immunization programs in developed countries, pertussis was one of the most common infectious diseases for morbidity and mortality. Due to the promotion and coverage of pertussis vaccines, the incidence of whooping cough worldwide has declined, but in recent years the incidence of pertussis has shown a significant upward trend, and the incidence of pertussis in children has increas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2531/119C12Q2522/101Y02A50/30
Inventor 许腾曾伟奇杨敏玲徐学中刘足李永军王小锐苏杭
Owner 广州微远医疗器械有限公司
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