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Primer, probe, reagent and method for rapidly detecting feline parvoviruses at normal temperature and constant temperature

A feline parvovirus, normal temperature and isothermal technology, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of expensive equipment, time-consuming, unpopularization, etc., to prevent false positive interference , the effect of preventing pollution

Inactive Publication Date: 2020-10-02
潍坊安普未来生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, most of the diagnostic methods for feline parvovirus are serological methods and virus isolation and identification, which have low sensitivity and cannot diagnose early infection; while PCR-based diagnostic methods are time-consuming, require high technical requirements, and expensive equipment, so they cannot be popularized
[0004] In summary, there are obviously inconveniences and defects in the actual use of the existing technology, so it is necessary to improve

Method used

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  • Primer, probe, reagent and method for rapidly detecting feline parvoviruses at normal temperature and constant temperature
  • Primer, probe, reagent and method for rapidly detecting feline parvoviruses at normal temperature and constant temperature
  • Primer, probe, reagent and method for rapidly detecting feline parvoviruses at normal temperature and constant temperature

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Screening of primers

[0050] With reference to the Feline panleukopenia virus (FPV) VP2 gene sequence published on GenBank, the inventor has conducted a more in-depth study on the genome, protein structure and function of the VP2 of FPV, and the content of the VP2 gene of FPV Higher. The present invention selects the VP2 gene of FPV as the target gene. A large number of experiments show that different primers have a certain influence on the effect and sensitivity of constant temperature amplification. Therefore, in this study, four pairs of different primers were initially designed for the VP2 gene of FPV: VP-1, VP-2, VP-3 and VP-4 (as shown in Table 1). These sequences can be compared with the VP2 gene of FPV. The corresponding sequence specifically binds.

[0051]

[0052]

[0053] Table 1: Sequences of primers and probes

[0054] image 3 It is the electrophoresis pattern of the primer's influence on the amplification effect. In the figure, M is the Marker,...

Embodiment 2

[0059] Example 2: Fluorescence detection

[0060] This embodiment is used to illustrate the normal temperature and constant temperature fluorescence reaction performed on a fluorescent instrument.

[0061] 1. According to the feline parvovirus VP2 gene sequence published on NCBI GenBank, the feline parvovirus VP2 gene sequence is fully synthesized.

[0062] 2. Feline parvovirus VP2 gene sequence designed a pair of primers (VP2-F1 and VP2-R1) and a fluorescent probe (VP2-Probe), the sequence is shown in Table 2 below:

[0063]

[0064] Table 2 Sequences of primers and probes of feline parvovirus VP2 gene

[0065] In Table 2, (F) is Fluorophore, (H) is THF residue, (B) is Quencher, and the 3'end is labeled with Biotin-TEG.

[0066] 3. The amplification reaction system is:

[0067]

[0068] Remarks: Lyophilized powder reagent contains the necessary enzymes and auxiliary components mentioned above.

[0069] 4. Amplification reaction program: a constant temperature of 39 degrees, 20 minutes.

[...

Embodiment 3

[0071] Example 3: Sample detection

[0072] In order to test the specificity of this detection method, samples of cats often suffering from different viruses are collected for testing.

[0073] The test results showed that only the feline parvovirus samples showed positive results of normal amplification, and none of the feline coronavirus samples, feline herpesvirus type I samples and negative controls showed amplification. Figure 5 As shown, 1 and 2 are feline parvovirus samples, 3 are feline coronavirus samples, 4 are feline herpes virus type I samples, and 5 are negative control groups. The above results indicate that the MIRA constant temperature fluorescence detection kit of the present invention can specifically amplify the target sequence in FPV without cross-reacting with other viral nucleic acids. It shows that the method of the invention has good specificity and no false negatives.

[0074] In summary, the present invention can perform rapid, real-time, and specific det...

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Abstract

The invention is applicable to the technical field of feline parvovirus detection, and provides a primer, a probe, a reagent and a method for rapidly detecting feline parvoviruses at a normal temperature and a constant temperature. According to the invention, the feline parvoviruses can be rapidly and specifically detected in real time; under the combined action of a specific primer, a specific fluorescent probe, five engineering enzymes and chemical components, rapid detection of a nucleic acid target object is achieved in an instrument with a fluorescence detection function; meanwhile, closed tube detection is guaranteed through strict experiment operation steps; the aerosol pollution is effectively prevented; and the primer, probe, reagent and method provided by the invention are applicable to being applied to various fluorescence detection devices for detection.

Description

Technical field [0001] The invention relates to the technical field of feline parvovirus detection, in particular to a primer, probe, reagent and method for rapid detection of feline parvovirus at room temperature and isothermally. Background technique [0002] Feline panleukopenia virus (Feline panleukopenia virus), also known as feline panleukopenia virus and cat plague virus, is a single-stranded DNA virus belonging to the Parvoviridae family and the genus Parvovirus. It can cause loss of appetite, high fever, lethargy, runny nose, dehydration and vomiting in cats. , Severe reduction of white blood cells and symptoms such as hemorrhagic enteritis. Feline parvovirus does not infect humans, but it can survive in the environment for up to one year. Therefore, cats are easily exposed to the virus and become infected, especially kittens are extremely susceptible to infection. The infection rate of cats under 1 year old can be Up to 70%, the mortality rate is 50% to 60%, and the mo...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2563/107
Inventor 刘琼瑶宋文凤娄志英刘国宪
Owner 潍坊安普未来生物科技有限公司
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