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Composition, kit and application for detecting feline calicivirus and feline parvovirus

A feline calicivirus and feline parvovirus technology, applied in the field of genetic engineering, can solve problems such as low sensitivity and long reaction time, achieve high sensitivity, reduce detection costs, and avoid false positives

Active Publication Date: 2020-01-03
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the defects in the prior art that the detection of FCV adopts single-channel fluorescent RT-PCR method or fluorescent RT-PCR has low sensitivity, long reaction time, or no detection of FPV fluorescent RT-PCR method, etc., this paper The invention provides a composition, kit and application for detecting feline calicivirus (FCV) and feline parvovirus (FPV), which detects amplification products in a closed-tube state, avoiding contamination caused by amplification products False positive; probe hybridization specificity is stronger; the detection of FCV and FPV can be realized at the same time, which greatly shortens the detection time and reduces the detection cost; there is no need for subsequent processing after PCR, and the operation is simpler and faster, safe and pollution-free

Method used

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  • Composition, kit and application for detecting feline calicivirus and feline parvovirus
  • Composition, kit and application for detecting feline calicivirus and feline parvovirus
  • Composition, kit and application for detecting feline calicivirus and feline parvovirus

Examples

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Embodiment 1

[0065] This embodiment is a design of specific primers and probes for the detection of feline calicivirus (FCV) and feline parvovirus (FPV).

[0066] Primers and probes were designed according to the conserved sequence of the FCV ORF1 gene, and then primers and probes were designed according to the conserved sequence of the FPV VP2 gene, as shown in Table 1 below. According to the following sequences, specific fluorescent RT-PCR primers and probes were synthesized. with DEPC-H 2 O dilute the primers to 10 μM and store at -20°C for later use.

[0067] Table 1 Primer and Probe Combinations

[0068]

[0069] Wherein the nucleotide sequence of FCV-F1 is shown in SEQ ID NO.1 in the sequence listing, the nucleotide sequence of FCV-R1 is shown in SEQ ID NO.2 in the sequence listing, and the nucleotide sequence of FPV-F1 is shown in Shown in SEQ ID NO.3 in the sequence listing, the nucleotide sequence of FPV-R1 is shown in SEQ ID NO.4 in the sequence listing, and the nucleotide s...

Embodiment 2

[0071] This example is the establishment and optimization of a preferred reaction system containing combination 1 in Example 1 for the detection of feline calicivirus (FCV) and feline parvovirus (FPV).

[0072] 1. Sample preparation: Synthesize the target amplification regions FCV ORF1 and FPV VP2, connect to the PET30a vector, transform into BL21(B) recipient bacteria, select positive clones, and construct positive plasmids containing the target amplification regions of FCV and FPV as Positive controls for FCV and FPV detection; RV vaccine strains, FHV vaccine strains, FIPV vaccine strains, CIV positive samples, CDV positive samples, etc. TRIZOL extracts the nucleic acid of the above positive control and specific reference product, and the negative control is ready for use.

[0073] 2. Optimization of the concentration of primers and probes: In the case of other components in the reaction system remaining unchanged, PCR reactions were performed using primers and probes from 1...

Embodiment 3

[0081] This embodiment is a preferred establishment of a standard curve using combination 1 in embodiment 1.

[0082] After measuring the concentration of FCV and FPV positive plasmid DNA with a spectrophotometer, use DEPC-H 2 O Make a 10-fold serial dilution of the plasmid standard so that the copy number in each 5 μL detection volume is 5.5×10 7 , 5.5×10 6 , 5.5×10 5 , 5.5×10 4 , 5.5×10 3copy / μL, set 3 repetitions, and perform amplification. After the reaction, the standard curve was automatically obtained using the Vii fluorescent quantitative PCR analysis software. The results showed that the FAM detection channel for detecting FCV established with primers (FCV-F1 and FCV-R1) and probe FCV-P1 showed a typical S-type amplification curve with obvious exponential area. The values ​​presented have a good linear range with a slope close to -3.523 and a correlation coefficient of R 2 is 0.998 (see figure 1 with figure 2 ). The HEX detection channel for detecting FPV es...

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Abstract

The invention relates to a composition for detecting feline calicivirus and feline parvovirus. The composition comprises primers and probes for detecting the feline calicivirus and the feline parvovirus respectively, wherein the primers include, for example,the sequences shown in SEQ ID NO.1 to SEQ ID NO.4, the probes include, for example,the sequences shown inSEQ ID NO.5 to SEQ ID NO.6; or, the primers include, for example, the sequences shown inSEQ ID NO.7 to SEQ ID NO10, the probes include, for example, the sequences shown inSEQ ID NO.11 to SEQ ID NO12. The invention further relates to a kit and application comprising the composition. The kit performs amplification reaction and product detection in a closed tube state, and false positivity due to contamination of amplification productsis avoided; the probes are higher in hybridization specificity and higher in sensitivity; and no follow-up treatment is after the PCR, operation is simpler and faster, safe and pollution-free effectsare realized, and the detection of both FCV and FPV viruses can be realized at the same time.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a composition, a kit and an application thereof for detecting feline calicivirus (FCV) and feline parvovirus (FPV). Background technique [0002] Feline calicivirus (FCV) is a member of the genus Vesivirus in the family Calicivirdae, and is a highly pathogenic pathogen of most cats. It is widely prevalent in cats and is distributed worldwide. FCV mainly infects cats and can cause typical respiratory symptoms and oral ulcers. In addition, it can also infect wild cats including lions, tigers, and cheetahs. The health of cats and rare wild animals poses a great threat. In recent years, feline malignant systemic disease (FCV-associateed virulent systemic disease) caused by FCV infection has broken out successively in China, the United States, the United Kingdom, Italy and other countries. This strain is called FCV-VSD strain, which can infect adult cats and It causes sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2563/107C12Q2521/107Y02A50/30
Inventor 沈海潇鞠厚斌杨德全赵洪进王建李鑫杨显超葛菲菲刘健邓波
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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