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Kit for detecting FCV, FPV and FHV-1 viruses by nucleic acid extraction-free triple fluorescence RT-LAMP

A technology of RT-LAMP and FHV-1, which is applied in the direction of microbe-based methods, microbiological measurement/testing, microbiology, etc., and can solve problems such as false positives of primers

Pending Publication Date: 2021-09-14
苏州艾可瑞动物检测技术服务有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the existence of a variety of target gene-specific primers in the multiple LAMP reaction system, there may be interference between primers or pairing to cause non-specific amplification reactions, resulting in false positives

Method used

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  • Kit for detecting FCV, FPV and FHV-1 viruses by nucleic acid extraction-free triple fluorescence RT-LAMP
  • Kit for detecting FCV, FPV and FHV-1 viruses by nucleic acid extraction-free triple fluorescence RT-LAMP
  • Kit for detecting FCV, FPV and FHV-1 viruses by nucleic acid extraction-free triple fluorescence RT-LAMP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Primer Design and Synthesis

[0064] For the conserved sequences of the FCV ORF2 gene, FPV VP2 gene, and FHV-1NK gene, multiple sets of LAMP primer pairs were automatically generated using the online software PrimerExplorer V5 (http: / / primerexplorer.jp / e / index.html) and screened to obtain the FCV LAMP primer pairs of ORF2 gene, FPV VP2 gene, FHV-1NK gene: F3, B3, FIP, BIP, LF, LB. In addition, the assimilation probe primers were designed, including fluorescent strand (Fluorescent strand) F strand and quenching strand (Quench strand) Q strand. The 3' end of the fluorescent strand is a forward loop primer (LF) sequence, and its 5' end sequence is complementary to the quenched strand. The 5' end of the FCV fluorescent chain primer is labeled with a FAM fluorescent group, the FPV assimilation probe is labeled with a VIC fluorescent group at the 5' end of the F chain, and the FHV-1 assimilation probe is labeled with a NED fluorescent group at the 5' end of the F c...

Embodiment 2

[0070] Example 2 Nucleic acid extraction-free (RT)-LAMP reaction system and thermal cracking temperature optimization

[0071] Take 100 μL of vaccine stock solution (Miaosanduo inactivated vaccine was purchased from Zoetis (Shanghai) Enterprise Management Co., Ltd., production batch number: D216520A) with a gradient of 3 °C, using different cracking temperatures (93 °C, 90 °C, 87 °C, 84 °C, °C) in a water bath for 5 minutes, and on ice for 3 minutes, absorb 5 μL of the supernatant as an amplification template, and use nuclease-free sterilized water as a negative control. The conventional (RT)-LAMP reaction of the three viral genes was carried out according to the following reaction system and reaction procedure.

[0072] Table 2 (RT)-LAMP reaction liquid preparation method

[0073]

[0074] Gently mix the above reaction solution on ice, and set up a negative control with nuclease-free sterilized water. After reacting in a PCR instrument at 65°C for 60 minutes, carefully ab...

Embodiment 3

[0076] Example 3 Primer concentration and multiple fluorescent RT-LAMP system

[0077]Single (RT)-LAMP primer mixture (primer mix, PM) is composed of: FIP / BIP Primers 1.6 μM; F3 / B3 Primers 0.2 μM; LF / LB Primers 0.2 μM; F strand 0.1 μM, mix the above components, The concentration of a set of primers for a single target gene in the 25 μL reaction system was set to 4.1 μmol / L. Since the assimilative probe primer is quoted, the quenching strand (Quench strand) Q strand in the single-plex fluorescent (RT)-LAMP reaction is the same, so the final concentration of the quenching primer in the multiple fluorescent RT-LAMP 25μL reaction system only needs 0.2 μM. Multiple fluorescent RT-LAMP needs to optimize the ratio of mixed primers, so the primers with different concentrations are combined according to the following methods:

[0078] (1) The primer concentrations of FCV, FPV and FHV-1 were all set at 4.1 μmol / L.

[0079] (2) The primer concentrations of FCV, FPV and FHV-1 were all ...

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Abstract

The invention discloses a kit for detecting FCV, FPV and FHV-1 viruses by nucleic acid extraction-free triple fluorescence RT-LAMP. An RT-LMAP detection method which is high in sensitivity, high in specificity and good in repeatability, does not need to extract nucleic acid of a detection sample and can simultaneously detect the FCV, the FPV and the FHV-1 viruses by directly adopting a clinical sample stock solution is established by utilizing an LAMP technology, and a rapid detection kit is developed. The nucleic acid extraction-free triple fluorescence RT-LAMP detection method established by the test and the developed detection kit have good specificity and sensitivity and good stability, only the thermal cracking clinical sample stock solution is used, nucleic acid extraction is not needed, detection of related pathogens can be completed within 1h, and the method is an effective method for clinically and rapidly detecting the FCV, the FPV and the FHV-1 viruses.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and relates to a nucleic acid extraction-free triple fluorescent RT-LAMP detection kit for FCV, FPV and FHV-1 viruses. Background technique [0002] Feline calicivirus (Feline calicivirus, FCV), feline parvovirus (Feline panleukopeniavirus, FPV) and feline herpesvirus-1 (Feline herpesvirus-1, FHV-1) are the three main infectious diseases that endanger the health of feline animals clinically. The virus is highly contagious and has a high fatality rate. Their common laboratory detection methods mainly include electron microscope observation, PCR, fluorescence quantitative PCR (Real-time quantitative PCR, qPCR), recombinase polymerase amplification (Recombinase polymerase amplification, RPA), enzyme-linked immunosorbent assay (Enzyme linkedimmunosorbent assay, ELISA), colloidal gold immunochromatography, etc. Due to the shortcomings of these methods, such as high requirements on exper...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/701C12Q1/705C12Q1/6844C12Q2600/16C12Q2531/119C12Q2537/143C12Q2563/107Y02A50/30
Inventor 沈新何成华何力力
Owner 苏州艾可瑞动物检测技术服务有限公司
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