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Triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application

A fluorescence quantitative and detection reagent technology, applied in the field of pathogen detection, to achieve the effect of low cost, high sensitivity and improved work efficiency

Active Publication Date: 2021-07-13
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem that the three pathogens of SARS-CoV-2, IAV and FCV cannot be distinguished by clinical symptoms in pets, and hope to develop a detection method that can diagnose these three pathogens at one time, the present invention provides a triple TaqMan Fluorescent quantitative PCR detection reagents, kits, detection methods and applications. Compared with ordinary PCR, this detection method has the advantages of high sensitivity, strong specificity, short detection cycle, and low labor cost required for detection. It is especially suitable for large-scale detection and mixed infection

Method used

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  • Triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application
  • Triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application
  • Triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application

Examples

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Effect test

Embodiment 1

[0044] A triple TaqMan fluorescent quantitative PCR detection reagent, including specific qPCR detection primers for novel coronavirus (SARS-CoV-2), feline calicivirus (FCV), influenza A (influenza A virus, IAV) and The corresponding TaqMan probes are as follows:

[0045] (1) The primer sequence and TaqMan probe sequence used to detect SARS-CoV-2 are: upstream primer N-112F, downstream primer N-112R, TaqMan probe N-probe;

[0046] The N-112F has a sequence as shown in SEQ ID No.1

[0047] The N-112R has a sequence as shown in SEQ ID No.2;

[0048] The N-probe has a sequence as shown in SEQ ID No.3;

[0049] The 5' end of the probe N-probe is labeled with a reporter fluorescent dye of HEX, and the 3' end of which is marked with a fluorescent quencher group of BHQ1: 5'-HEX-TTCACCGCTCTCACTCAACAT-BHQ1-3'.

[0050] (2) The primer sequence and TaqMan probe sequence used to detect FCV are: upstream primer FCV-106F, downstream primer FCV-106R, TaqMan probe FCV-probe;

[0051] The ...

Embodiment 2

[0063] Based on the triple fluorescent quantitative qPCR detection method of the detection reagent described in Example 1, the specific steps are as follows:

[0064] (1) Obtain the cDNA of the sample to be tested: extract the total RNA of the cat nasopharyngeal swab sample or respiratory tissue or organ, and reverse transcribe to obtain the cDNA template.

[0065] The reverse transcription was carried out using HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper). The total reaction system was 20 μL, including 1 μL Random hexamers, 6 μL RNase-free Water, 5 μL total RNA, and heated at 65°C for 5 min after mixing, and quickly Place it on ice for quenching, and let it stand on ice for 2 minutes, then continue to add 4×gDNA wiperMix, 4 μL, then gently blow and mix with a pipette, 42°C for 2 minutes; finally add 2 μL 10×RT Mix, 2 μL HiScriptII Enzyme Mix , after mixing, 5min at 25°C, 45min at 50°C, and 2min at 85°C to get it.

[0066] (2) Three sets of qPCR primers and probes ...

Embodiment 3

[0071] The construction and verification of the reagent described in Example 1 and the detection method described in Example 2, the specific experimental process is as follows:

[0072] 1. Establishment of standard curve

[0073] (1) Design of primers and probes: All probes and primers in this experiment were designed using the primer design software Oligo 7, and were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0074] (2) Preparation of plasmid standards: Using the primers N-112F (F / R), FCV-106F (F / R), and IAV-149F (F / R) designed in this experiment, the high-fidelity PCR enzyme Phanta Max Super- Fidelity DNA Polymerase (Nanjing Novizan Biotechnology Co., Ltd.) was used to amplify the target fragment, and then the target fragment was connected to the pMD18-T vector (Bao Bioengineering (Dalian) Co., Ltd.) vector to construct a recombinant plasmid. The reaction system and experimental program settings of the PCR amplification reaction were set according to the inst...

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Abstract

The invention discloses a triple TaqMan fluorescent quantitative PCR detection reagent, a kit, a detection method and application. The detection reagent comprises a specific qPCR detection primer for a novel coronavirus (SARS-CoV-2), a feline calicivirus (FCV) and an influenza A virus (IAV), and a corresponding TaqMan probe. According to the detection method, three pairs of specific primers and TaqMan probes are designed mainly aiming at SARS-CoV-2, FCV and IAV, three kinds of cat respiratory viruses can be simultaneously detected in one PCR reaction tube, the detection sensitivity can reach 1 * 10 < 1 > copies / mu L, and the detection method has excellent specificity and repeatability and can be applied to clinical detection of pets.

Description

technical field [0001] The invention belongs to the technical field of pathogen detection, and in particular relates to a triple TaqMan fluorescent quantitative PCR detection reagent, kit, detection method and application. Background technique [0002] As a companion animal, cats are closely related to the daily life of humans, and current studies have shown that SARS-CoV-2 can infect cats and can be transmitted between cats, although there is no evidence that SARS-CoV-2 It can be transmitted from cats to humans, but as mentioned earlier, the phenomenon of cross-host transmission is visible in coronaviruses. In order to strictly prevent and control COVID-19, timely monitoring of SARS-CoV-2 in cats It is extremely necessary to isolate positively infected cats. [0003] Influenza A virus, as a common influenza virus, is highly pathogenic to humans and has caused many worldwide pandemics. From 1918 to 1920, the H1N1 pandemic killed about 20 million people. Influenza A also b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143C12Q2561/101Y02A50/30
Inventor 粟硕张文艳梁家玮李改茹
Owner NANJING AGRICULTURAL UNIVERSITY
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