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Primer pairs for fluorescence EMA detection of canine distemper virus, kit and detection method

A technology of canine distemper virus and detection kit, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of long time-consuming, expensive and unusable virus isolation and culture, and achieve results The report is clear, the detection is simple and easy, and the effect on detection ability

Inactive Publication Date: 2018-04-17
SUZHOU CLICKGENE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional detection methods for canine distemper mainly include virus isolation and culture, ELISA detection, immune test strip detection and RT-PCR detection, etc. There are problems such as low sensitivity or low specificity. At present, many patents use RT-PCR or LAMP methods. These methods have high sensitivity and specificity, but require expensive instrument reagents and require high technical requirements for operators. At the same time, it takes a long time, so it cannot be used for grassroots and on-site inspections

Method used

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  • Primer pairs for fluorescence EMA detection of canine distemper virus, kit and detection method
  • Primer pairs for fluorescence EMA detection of canine distemper virus, kit and detection method
  • Primer pairs for fluorescence EMA detection of canine distemper virus, kit and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, canine distemper virus fluorescent EMA detects

[0063] Canine distemper virus was detected by fluorescent EMA detection kit. The canine distemper virus fluorescence EMA detection kit comprises:

[0064] 1. 500 μl of complex solution;

[0065] Each 30ml reconstitution solution contains:

[0066]

[0067] 2. 48 pieces of freeze-dried enzyme tubes containing primers;

[0068] Each 4mL freeze-dried mixture contains the following components:

[0069]

[0070]

[0071] Wherein, the sequences of primer pairs and probes are as follows:

[0072] Upstream primer: 5'-AAATCTGGCTTGATATTGTTAG-3';

[0073] Downstream primer: 5'-GAGCCGGATACATAGTTTCAA-3';

[0074] Probe: 5'-CCCCAGGGAACAAGCCTAC / i6-FAMdT / GA / idSp / A / iBHQ1dT / GATTTGT-3' C3Spacer.

[0075] 3. The negative control substance is the RNase-free water blank control.

[0076] 4. The positive control substance is canine distemper pseudovirus, the concentration is 1 μg / mL.

[0077] Canine distemper vir...

Embodiment 2

[0083] Embodiment 2: the detection of clinical sample:

[0084] Canine distemper virus fluorescent EMA detection kit was used to detect canine distemper virus clinical samples. The canine distemper virus fluorescence EMA detection kit comprises:

[0085] 1. 500 μl of complex solution;

[0086] Each 30ml reconstitution solution contains:

[0087]

[0088] 2. 48 pieces of freeze-dried enzyme tubes containing primers;

[0089] Each 4mL freeze-dried mixture contains the following components:

[0090]

[0091]

[0092] Wherein, the sequences of primer pairs and probes are as follows:

[0093] Upstream primer: 5'-AAATCTGGCTTGATATTGTTAG-3';

[0094] Downstream primer: 5'-GAGCCGGATACATAGTTTCAA-3';

[0095] Probe: 5'-CCCCAGGGAACAAGCCTAC / i6-FAMdT / GA / idSp / A / iBHQ1dT / GATTTGT-3' C3Spacer.

[0096] 3. The negative control substance is the RNase-free water blank control.

[0097] 4. The positive control substance is canine distemper pseudovirus, the concentration is 1 μg / mL.

...

Embodiment 3

[0100] Embodiment 3: Specific detection:

[0101] With canine parvovirus, canine adenovirus type 1, canine adenovirus type 2, canine parainfluenza virus, canine leptospira, canine coronavirus nucleic acid, with canine distemper virus positive samples as contrast, the specificity detection of the present invention is carried out, The results show that only the positive samples of canine distemper virus are positive results, while other samples are horizontal lines, which are negative results, which proves that the present invention is compatible with canine parvovirus, canine adenovirus type 1, canine adenovirus type 2, canine parainfluenza virus, canine There was no cross-reaction between Leptospira and canine coronavirus.

[0102] The invention uses normal temperature nucleic acid amplification technology, through multiple enzymatic reactions such as M-MLV reverse transcriptase, RNase inhibitor, DNA helicase, single-stranded DNA binding protein, DNA polymerase, etc. In 10-30...

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Abstract

The invention relates to primer pairs for fluorescence EMA detection of canine distemper virus (CDV), a kit and a detection method. The primer pairs for fluorescence EMA detection of canine distempervirus (CDV) contain a forward primer, a reverse primer and a probe. The kit comprises the following contents: a redissolved solution, a lyophozyme tube containing primers, a positive quality control and a negative quality control. The detection includes the process of carrying out a normal-temperature nucleic acid amplification technology by using the kit. Through multiple enzymatic reactions of M-MLV reverse transcriptase, a Rnase inhibitor, DNA helicase, single-stranded DNA binding protein, DNA polymerase and the like, RNA to be tested can undergo reverse transcription into cDNA under the condition of constant temperature 37-45 DEG C within 10-30 min, and the cDNA is amplified by millions of times; and and with the cooperation of the fluorescence detection technique, rapid detection of RNA to be tested can be realized. The detection method of the invention has advantages of simple operation, short time and low requirement on equipment, and is suitable for rapid diagnosis of pet infectious diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fluorescent EMA detection primer set, a kit and a detection method for canine distemper virus. Background technique [0002] Canine distemper (Canine Distemper) is a highly contagious disease caused by canine distemper virus (Canine Distemper virus, CDV). According to the statistics of the list of animal diseases released on December 11, canine distemper belongs to the third category of animal diseases, mainly infecting dogs and cats. The transmission channel is mainly through direct contact with sick animals, and can also be transmitted through air or food. Belongs to zoonotic diseases. [0003] Under natural conditions, canine distemper can infect dogs of different ages, sexes and breeds, but it is the most susceptible to juvenile puppies. Other studies have shown that in addition to canids, many animals such as mustelidae, raccoons, and giant pandas can also be infected. It is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2527/127C12Q2521/107C12Q2521/513C12Q2521/101C12Q2563/107C12Q2522/101
Inventor 谭卓胡振新郜安国
Owner SUZHOU CLICKGENE BIOTECH CO LTD
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