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Real-time fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detection of foot and mouth disease virus and seneca valley virus and application

A real-time fluorescence quantitative and foot-and-mouth disease virus technology, applied in the field of molecular biology detection, can solve the problems of not being able to adapt to the rapid, sensitive and accurate quantitative detection of foot-and-mouth disease virus and Seneca Valley virus, etc., and achieve low cost, good specificity, The effect of shortening the reaction time

Inactive Publication Date: 2018-08-10
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the shortcoming that the prior art cannot adapt to the rapid, sensitive, accurate and quantitative detection of foot-and-mouth disease virus and Seneca Valley virus, thereby providing a method that can quickly, sensitively, accurately and quantitatively detect foot-and-mouth disease virus and The Taqman real-time fluorescent quantitative RT-PCR kit of Seneca Valley virus, the present invention also provides the use method of this kit

Method used

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  • Real-time fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detection of foot and mouth disease virus and seneca valley virus and application
  • Real-time fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detection of foot and mouth disease virus and seneca valley virus and application
  • Real-time fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detection of foot and mouth disease virus and seneca valley virus and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Design and preparation of embodiment 1 primers and probes

[0039] Refer to GenBank (gene bank) to find SVV strains and FMDV strains, find the conserved regions on each sequence through sequence comparison, select the conserved regions to design and screen a pair of amplification primers and a probe primer, the sequence is as follows :

[0040] The primer sequences for amplifying SVV strains are:

[0041] Upstream primer: 5'-GGCCGCCACGCTATCTAACCAA-3' (shown in SEQ ID NO.1),

[0042] Downstream primer: 5'-CACGGGCCCGAGCTTCTTCATC-3' (shown in SEQ ID NO.2).

[0043] The probe primer sequence is: 5'-CTTCAGTGAAAGCTC-3' (shown in SEQ ID NO.5).

[0044] The primer sequences for amplifying the FMDV strain are:

[0045] Upstream primer: 5'-TCCGGACCAGACGAGTA-3' (shown in SEQ ID NO.3),

[0046] Downstream primer: 5'-CCCAACGCAGGTAGAGTG-3' (shown in SEQ ID NO.4).

[0047] The probe primer sequence is: 5'-CGGCGTCTCTTTGA-3' (shown in SEQ ID NO.6).

[0048] The above primers and ...

Embodiment 2

[0049] Example 2 Assembly of one-step direct amplification Taqman real-time fluorescent quantitative RT-PCR kit for detecting foot-and-mouth disease virus and Seneca Valley virus

[0050] 1. The primer shown in SEQ ID NO.1-4 prepared in Example 1 and the probe shown in SEQ ID NO.5-6 are mixed according to the molar ratio 1:1:1:1:1:1, and the primer And the concentration of the probe is 10 pmol / μl.

[0051] 2. Nucleic acid extraction solution:

[0052] Dissolve 25g of guanidine isothiocyanate in 33ml of CSB buffer and mix until completely dissolved. Wherein the CSB buffer contains: 42mM sodium citrate; 0.83w / v% N-lauryl sarcosine (sodium lauryl sarcosine); 0.2mM β-mercaptoethanol.

[0053] 3. 2×Direct qRT-PCR Mix

[0054] 4. Mixed enzyme solution

[0055] 5. Standard positive plasmid

[0056] The standard positive plasmid was prepared according to the following steps:

[0057] a. Design a pair of amplification primers SVV-F, SVV- R, FMDV-F, and FMDV-R, the amplified frag...

Embodiment 3

[0067] Embodiment 3 The application of Taqman real-time fluorescent quantitative RT-PCR kit of the present invention in detecting FMDV and SVV

[0068] 1. Preparation of standard curve

[0069] a. Perform a 10-fold dilution of the pMD-397 and pMD-109 standard positive plasmids to make it: 10 -1 -10 -15 , and quantitative PCR was performed in triplicate for each gradient.

[0070] b. According to the amplification situation, select 5-6 points to make a standard curve suitable for the standard curve requirements. Such as figure 1 shown.

[0071] 2. Sample detection

[0072] (1) The total PCR system is 50 μl:

[0073] a. 2×Direct qRT-PCR Mix: 37 μL; b. Mixed enzyme solution: 2 μL; c. Mixed solution of primers and probes with a concentration of 10 pmol / μl: 6 μL. Add the above components to 0.2ml amplification in turn tube;

[0074] (2) Add 5 μl of vesicle fluid from the pig’s trotters to be tested to the above-mentioned amplification tube, centrifuge at 12000 rpm for 5-30 ...

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Abstract

The invention discloses a real-time fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) kit for detection of foot and mouth disease virus and seneca valley virus and application. The kit comprises primers and probes for detecting the foot and mouth disease virus and the seneca valley virus and preferably further comprises nucleic acid extract liquid, 2*Direct qRT-PCRMix, enzyme mixed liquid, negative control and positive control. By adoption of the kit for detecting the foot and mouth disease virus and the seneca valley virus, high specificity, high sensitivity,high stability, simplicity and convenience in operation and the like are achieved. Without extra extraction of virus RNA and reverse transcription, a user only needs to add a to-be-tested sample intoa reaction tube, then performs quantitative analysis on a start template according to fluorescence signal changes and a Ct value and standard curve relation and finally calculates a copy number of the to-be-tested sample. The kit is not only applicable to quantitative analysis in research and development institutions but also suitable for pathogen detection and analysis in all levels of prevention and control institutions, basic veterinary stations, large and medium sized farms and the like, thereby having a promising application prospect.

Description

technical field [0001] The present invention relates to a kind of virus detection kit and application thereof, particularly a kind of one-step direct expansion Taqman real-time fluorescent quantitative RT-PCR (probe method real-time-quantitative polymerase chain) for detecting foot-and-mouth disease virus and Seneca Valley virus simultaneously Reaction) kit, the present invention also relates to the application of this kit in detecting foot-and-mouth disease virus and Seneca Valley virus. The invention belongs to the technical field of molecular biology detection. Background technique [0002] Foot and Mouth Disease Virus (FMDV) and Senecavalley virus (SVV) infect pigs can cause primary vesicular disease in pigs. The clinical symptoms are similar and the two cannot be distinguished. SVV has successively launched in the United States (2015, 2016, 2017), Canada (2007, 2011, 2015, 2016), Brazil (2014, 2015), Colombia (2016), China (2015, 2016, 2017) and Thailand (2016) and ot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 郑海学田宏杨帆石正旺朱紫祥李丹张克山曹伟军刘永杰郭建宏何继军马旭升茹毅李林林刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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