Fluorescent quantitative PCR detection primer for simultaneously detecting feline parvovirus and feline HIV, probe and kit thereof

A technology for feline parvovirus and HIV, applied in the field of molecular biology, to achieve the effects of avoiding omissions, good specificity, and high sensitivity

Pending Publication Date: 2021-02-23
HANGZHOU ALLTEST BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For its treatment, there is currently no effective vaccine, ...

Method used

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  • Fluorescent quantitative PCR detection primer for simultaneously detecting feline parvovirus and feline HIV, probe and kit thereof
  • Fluorescent quantitative PCR detection primer for simultaneously detecting feline parvovirus and feline HIV, probe and kit thereof
  • Fluorescent quantitative PCR detection primer for simultaneously detecting feline parvovirus and feline HIV, probe and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Fluorescent quantitative PCR method detects feline parvovirus and feline HIV

[0036] After comparing the nucleic acid sequences of the feline parvovirus NS1 gene and the feline HIV pol gene, it was found that the two had no homology and no crossover. Then its sequence was designed by Primer Premier 5 and BeaconDesigner to design primers and probes, and synthesized by Shanghai Sangon Bioengineering Co., Ltd. The sequences of the upstream primers, downstream primers and probes for detecting feline parvovirus are respectively shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.5. The sequences of the upstream primers, downstream primers and probes for detecting feline HIV are respectively shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.6.

[0037] SEQ ID NO.1: 5'-GGCAAGCAATCCTCAGAGTC-3'

[0038] SEQ ID NO.2: 5'-GAGTACTCCACGGTTCCAGT-3'

[0039] SEQ ID NO.3: 5'-ACTCCGGACGTAGTGGACCTTGC-3'

[0040] SEQ ID NO.4: 5'-TGTACTTCCAGTCGAGCTTCT-3'

[0041] SEQ ID NO.5: ...

Embodiment 2

[0048] Embodiment 2: Fluorescent quantitative PCR detection kit for detecting feline parvovirus and feline HIV

[0049] A kit for detecting feline parvovirus and feline HIV by fluorescent quantitative PCR includes nucleic acid extraction reagents and qPCR detection reagents. The nucleic acid extraction reagents include Proteinase K, Buffer AL, Buffer AW1, BufferAW2 and Buffer AE. qPCR detection reagents include 2×Premix ExTaq Probe qPCR 10μL (containing Takara ExTaqHS, Mg 2+ , dNTP Mixture, Tli RNaseH), upstream and downstream primers and probes each 0.5 μL (concentration is 10 μmol / L), ddH 2 O 5 μL, positive control (plasmid containing feline parvovirus and feline HIV target gene amplification sequence) and negative control (ddH 2 O).

Embodiment 3

[0050] Embodiment 3: the specificity test that adopts embodiment 1 system to carry out

[0051] Take the plasmid containing the amplified sequence of the target gene of feline parvovirus and feline HIV as the positive control, and take the feline calicivirus vaccine strain and feline herpes virus vaccine strain as the experimental group, extract DNA, and perform qPCR with specific probes and primers Amplify. The steps include: (1) DNA extraction: extract with QIAamp DNA Mini Kit and store at -80°C for later use. (2) qPCR amplification: the amplification volume is 20uL, including primers shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and such as SEQ ID NO.5 and SEQ ID NO.5 and SEQ ID NO. 0.5uL of each probe indicated by ID NO.6 (concentration is 10μmol / L), 2×Premix ExTaq Probe qPCR 10uL, ddH 2 O 5uL and sample 2uL. The qPCR amplification conditions were: preheating at 95°C for 30s, denaturation at 95°C for 10s, and annealing at 60°C for 20s as a cycle, a total o...

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Abstract

The invention discloses a fluorescent quantitative PCR detection primer for simultaneously detecting feline parvovirus and feline HIV, a probe and a kit . After the feline parvovirus NS1 gene sequenceand the feline HIV pol gene sequence are compared, it is found that the gene sequences of the feline parvovirus NS1 gene sequence and the feline HIV pol gene sequence do not intersect and are low inhomology. Therefore, a specific probe and a specific primer are designed according to the sequences of the feline parvovirus and the feline HIV, and the feline parvovirus and the feline HIV are diagnosed through fluorescent quantitative PCR amplification. The primer and the probe are prepared into a kit for simultaneously detecting the feline parvovirus and the feline AIDS virus. The whole processonly needs about 2 h, the time is short, the kit is easy to operate, high in sensitivity and good in specificity, the feline parvovirus and the feline AIDS virus can be detected at the same time, andomission of detection of one of the viruses can be avoided.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to fluorescent quantitative PCR detection primers, probes and kits for feline parvovirus and feline HIV. Background technique [0002] Feline parvovirus is also known as feline panleukopenia virus, feline distemper virus or feline infectious enteritis virus. It is mainly characterized by high fever, vomiting, severe leukopenia and enteritis. It is one of the main diseases that harm cats. In 1982, European and American scientists discovered feline parvovirus, and in 1957 Bilin isolated the virus for the first time, and then in 1964, Johnson also isolated the virus from the spleen of a leopard suspected of having symptoms of feline infectious enteritis. Feline parvovirus mainly infects the mucosal epithelial cells of the gastrointestinal tract, causing cell lesions and death. The clinical manifestations of feline panleukopenia caused by this virus can be divided ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/703C12Q1/701C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2563/107C12Q2537/143C12Q2545/113
Inventor 郭佳花孙晓笛陆维克陈金树
Owner HANGZHOU ALLTEST BIOTECH
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