Multiplex PCR rapid detection kit and primer group for common feline viruses

A detection kit and detection primer technology, which is applied in the field of cat respiratory and enterovirus nucleic acid detection, can solve public health threats and increase the risk of global pandemics, etc.

Pending Publication Date: 2021-06-08
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the canine and cat influenza virus gradually breaks through geographical barriers and spreads to all parts of the world through global trade, which increases the risk of a global pandemic and poses a potential threat to public health

Method used

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  • Multiplex PCR rapid detection kit and primer group for common feline viruses
  • Multiplex PCR rapid detection kit and primer group for common feline viruses
  • Multiplex PCR rapid detection kit and primer group for common feline viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1, kit assembly and use

[0026] To facilitate use, the reagent used in the present invention is assembled into four kinds of common viral multiple RT-PCR rapid detection kits, mainly composed of PCR standard, positive control, negative reference, primers, and casing. A corresponding container hole is provided in the box for each reagent.

[0027] The primers include the four pairs of PCR primers shown in Table 1 (Synthesis from Beijing Oyster New Industry Biotechnology Co., Ltd., the synthesis amount is 1OD per tube), which are common for FCV, FPV, FIV, FHV-1 four cats, They have the base sequence of sequence list seq.id.no.1 to seq.id.no.8, respectively. All upstream primers in PCR primers are co-packaging, all downstream primers.

[0028] Table 1 Multiple PCR primers designed in the present invention

[0029]

[0030] The primer design of the present invention is large and the existing PCR detection is large: on the one hand, due to the NS1 gene of cat films an...

Embodiment 2

[0042] Example 2, the present invention detects specific experiments of five viruses

[0043] According to the multi-PCR reaction system, Green Taq Mix (Vazyme) 14 μL, Fcv, FPV and FiV upper primer mixture 1.5 μL, downstream primer mixture 1.5 μL, FHV-1 upper prism, 0.75 μl, downstream primer, DDH 2 O 1 μL, 5.5 μl of sample DNA or cDNA template, including 1: ultrapure water negative control; 2: Fcv, FPV, FIV and FHV-1; 3: Fcv; 4: FPV; 5: FIV; 6: FHV- 1; 7: FCOV.

[0044] The reaction was carried out at the Life Proflex PCR amplifier, and the reaction procedure was: Pre-deformation of 95 ° C for 5 min, and the reaction conditions of 40 cycles were 95 ° C degeneration 35s, 56 ° C for 1 min, 72 ° C, and finally 72 ° C for 10 min. Take 10 μl of PCR product, in order to 2% agar gel electro-bore, 140V voltage, electrophoresis is about 20 min, and the photographic determination after the gel imager ultraviolet imaging, the results see Figure 5 The corresponding PCR product size is comple...

Embodiment 3

[0045] Example 3, the present invention detects sensitivity experiment of five viruses

[0046] Multi-PCR sensitivity detection reaction system: Green Taq Mix (Vazyme) 14 μL, Fcv, FPV and FIV upper primer mixture 1.5 μL, downstream primer mixture 1.5 μL, FHV-1 upper hopper primer 0.75 μL, downstream primer 0.75 μL, DDH 2 O1 μL, add 5.5 μL of 10 times series diluted FCV, FPV, FIV and FHV-1 yang-type mixtures (4.45 × 10 10 ~ 4.45 × 10 1 COPIES / μL) template.

[0047] The reaction was carried out at the Life Proflex PCR amplifier, and the reaction procedure was: Pre-deformation of 95 ° C for 5 min, and the reaction conditions of 40 cycles were 95 ° C degeneration 35s, 56 ° C for 1 min, 72 ° C, and finally 72 ° C for 10 min. Take 10 μl of PCR product, 2% agar gel electrophoresis.

[0048] The experimental results show that the minimum detection amount of multiple PCR reactions against FCV, FPV, FIV and FHV-1 virus is 24.48copies, which shows that the sensitivity of this detection met...

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Abstract

The invention discloses a multiplex PCR rapid detection primer group for common feline viruses, which comprises four pairs of PCR primers aiming at four feline respiratory tract and enteroviruses, namely FCV, FPV, FIV and FHV-1, and the four pairs of PCR primers respectively have base sequences from SEQ. ID. NO.1 to SEQ. ID. NO.8 in a sequence listing. Accordingly, a corresponding rapid detection kit is also developed. Single or mixed infection of four common feline viruses, namely FCV, FPV, FIV and FHV-1, can be simultaneously detected in the same reaction tube by using the rapid detection kit, the rapid detection kit has the characteristics of high specificity, good sensitivity, short time consumption and low cost, and the detection quantity of a recombinant plasmid standard substance can be as low as 4.45*10<1> copies / mu L. The multiplex PCR rapid detection kit and primer group provide quick and simple tools for detecting mixed infection of felines, lay a solid foundation for clinical quick identification and detection and laboratory epidemiological investigation, are beneficial to timely making a treatment scheme in pet diagnosis and treatment industry and reducing the death rate of the felines, and have important significance for public health and safety.

Description

Technical field [0001] The invention belongs to the field of cat respiratory tract and intestinal viral nucleic acid detection, and in particular, the present invention relates to a common detection kit with a common virus polymer PCR, and its primer group thereof. Background technique [0002] In recent years, with the rise of the pet industry, there are more common cases in a variety of pathogens in the cat population. Cat Cup Virus (FCV), Cat Tear Virus (FPV), Cat Inseparaminated Virus (FIV) and Cat Sperm Virus Type I Virus (FHV-1) is a major pathogens in the patriaries, due to their pathological changes in the clinic Symptoms and epidemiological similarities, it is difficult to distinguish these diseases. At the same time, the monitoring of the Cat Spled Virus I virus (FHV-1) is found in accordance with the inventors, which is more common in clinical cases, and the synergism of respiratory virus infections, the secondaryness will seriously endanger the health of cats. The abo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 陈樱郭嘉宁周华波郭金凡曾昊韦祖樟欧阳康黄伟坚
Owner GUANGXI UNIV
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