A primer set, reagent and method for detecting feline parvovirus based on polymerase helical reaction

A feline parvovirus, polymerase technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve strong chain displacement activity, shortened detection time, and good stress resistance.

Active Publication Date: 2022-06-14
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this series of PCR techniques cannot get rid of the limitation of reaction thermal cycles, and require complex temperature-variable instruments and skilled experimenters, resulting in high detection costs and long detection times

Method used

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  • A primer set, reagent and method for detecting feline parvovirus based on polymerase helical reaction
  • A primer set, reagent and method for detecting feline parvovirus based on polymerase helical reaction
  • A primer set, reagent and method for detecting feline parvovirus based on polymerase helical reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Primer Design

[0042] Use Oligo 6.0 software to design a pair of PCR primers for the conserved region of the FPV NS1 gene: FPV-F and FPV-R, the size of the amplified fragment is about 403bp, and design a pair of PSR-specific primers in the amplified fragment based on the PSR principle: PSR-FPVF, PSR-FPVR, and 1 pair of PSR accelerating primers: PSR-FPV-FJ, PSR-FPV-RJ. The PSR amplification product is a series of ladder-shaped bands. All primers were synthesized by Xi'an Branch of Beijing Qingke Biotechnology Co., Ltd. The specific primer names and sequences are listed in Table 1.

[0043] Table 1 PCR and PSR primers for detecting FPV

[0044]

[0045] 2. Nucleic acid extraction and copy number calculation

[0046] Use the viral nucleic acid extraction kit to extract viral nucleic acid from FPV positive samples, feline coronavirus (FCoV) positive samples, and feline norovirus (FNV) positive samples, respectively, use an ultramicro spectrophotometer to measure ...

Embodiment 2

[0055] In order to verify the influence of accelerating primers PSR-FPVF FJ and PSR-FPVF RJ on the experimental results, four sets of reaction systems were set up, the first group did not add any accelerating primers, the second group only added PSR-FPVF FJ, and the third group only added PSR -FPVF RJ, the fourth group joined both PSR-FPVF FJ and PSR-FPVF RJ. The detection method adopts the optimized result of Example 1.

[0056] see results figure 2 In the first group, a clear ladder-like band could be observed when the accelerated primer group was not introduced for 75 minutes; when the accelerated primer group was introduced, there was a PSR amplification product when the accelerated primer group was introduced for 45 minutes, but the fourth The electrophoretic bands of the first group were brighter than those of the second and third groups introduced with a single accelerated primer set at 45 minutes of reaction, indicating that the introduction of two accelerated primer...

Embodiment 3

[0058] With the FPV, FCoV and FNV genome nucleic acid that embodiment extracts as template, utilize the detection method of embodiment 1 and add acceleration primer PSR-FPVF FJ and PSR-FPVF RJ, carry out specificity test, set up simultaneously with sterilized water as template negative control.

[0059] see results image 3 , the results showed that no gene amplification occurred in the sterile water, FCoV and FNV groups, but gene amplification occurred in the FPV, FPV+FCoV, FPV+FNV, FPV+FCoV+FNV groups, see image 3 A. After the reaction, add a nucleic acid dye with a final concentration of 20×SYBRGreen I. Observe with the naked eye under visible light, and find that the three groups of sterilized water, FCoV and FNV are orange, FPV, FPV+FCoV, FPV+FNV, FPV+FCoV+FNV The color of the four groups changed from orange to green, which shows that the SYBR Green I staining results are consistent with the electrophoresis results, see image 3 B (Since the drawings in the description...

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Abstract

The invention discloses a primer set, a reagent and a method thereof for detecting feline parvovirus based on a polymerase helical reaction. The primer set includes specific primers PSR-FPVF and PSR-FPVR, accelerated primers PSR-FPVF FJ and PSR-FPVF RJ, the nucleotide sequence of the specific primer PSR-FPVF is shown in SEQ ID NO.1, the nucleotide sequence of the specific primer PSR-FPVR is shown in SEQ ID NO.2, and the nuclear sequence of the accelerated primer PSR-FPVF FJ The nucleotide sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the accelerating primer PSR‑FPVF RJ is shown in SEQ ID NO.4. The PSR detection method constructed by this primer set is simple to operate, has low requirements for detection equipment, and greatly shortens the detection time. It can be completed within 45 minutes at a constant temperature of 67°C. After the amplification, a final concentration of 20×SYBR is added to the system. Green I nucleic acid dye, the detection result can be judged by naked eyes under visible light, which greatly improves the detection efficiency.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer set, a reagent and a method for detecting feline parvovirus based on a polymerase helical reaction. Background technique [0002] Feline parvovirus (feline parvovirus, FPV), also known as feline panleukopenia virus or feline distemper virus, can cause an acute and highly contagious infectious disease in cats. FPV belongs to Parvoviridae, a single strand non-enveloped DNA virus. FPV can be transmitted directly through secretions such as saliva, and can also be transmitted indirectly through media such as clothes and shoes in indoor environments. Therefore, domestic cats are extremely susceptible to FPV infection, and the infection rate of young cats can be as high as 90%. The symptoms caused by FPV are very similar to those of other viral infections such as enteric coronavirus. Therefore, the establishment of a rapid, accurate, specific and sensitive detection ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
Inventor 魏衍全刘金波张勇薛惠文马永华杨孝朴武小椿
Owner GANSU AGRI UNIV
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