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Double microdroplet digital PCR (Polymerase Chain Reaction) absolute quantitative detection kit for porcine epidemic diarrhea virus and transmissible gastroenteritis virus

A porcine epidemic diarrhea and micro-droplet digital technology, which is applied in the determination/testing of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of complex test operation, long time required, low sensitivity, etc., and achieve dilution Template concentration, reduce the amount of template, ensure the effect of specificity

Inactive Publication Date: 2016-05-04
BEIJING SEAGULL BIOVENTURES & BIOTECH CO LTD
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

Traditional detection methods, such as virus neutralization test (VNT), immunofluorescence, immunoelectron microscopy, enzyme-linked immunosorbent assay, etc., take a long time to exist, rely on experienced testing personnel, complex test operations, and involve live virus operations , high requirements for the detection environment, and many shortcomings such as low sensitivity and poor repeatability
Although nucleic acid detection methods such as RT-PCR have the advantages of stronger specificity, higher sensitivity, higher degree of automation, and better repeatability of results than traditional methods, they can only achieve the detection of porcine transmissible gastroenteritis virus (TGEV). Qualitative and semi-quantitative detection of porcine epidemic diarrhea virus (PEDV), unable to perform accurate absolute quantitative detection of TGEV and PEDV nucleic acid, so the test results cannot reflect the real situation of virus infection in the sample
At the same time, the RT-PCR method still has certain limitations in sensitivity and specificity. It is more sensitive to certain chemical substances in the extraction process of viral nucleic acid, which in turn inhibits the PCR reaction, resulting in "false negative" results.
[0005] The concept of digital PCR (DigitalPCR, dPCR) was adopted and published by Bert Vogelstein as early as 1999. A small amount of mutant cells were produced, but because the consumables that could be used to dilute samples at that time were only 384-well plates, it was not very good to reflect the core concept of digital PCR - "terminal dilution" (terminal dilution)

Method used

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  • Double microdroplet digital PCR (Polymerase Chain Reaction) absolute quantitative detection kit for porcine epidemic diarrhea virus and transmissible gastroenteritis virus
  • Double microdroplet digital PCR (Polymerase Chain Reaction) absolute quantitative detection kit for porcine epidemic diarrhea virus and transmissible gastroenteritis virus
  • Double microdroplet digital PCR (Polymerase Chain Reaction) absolute quantitative detection kit for porcine epidemic diarrhea virus and transmissible gastroenteritis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 detects the design of specific primers and probes of porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus

[0044] 1. Design of primers and probes of the present invention: PEDV, GenBank accession number is AF353511, TGEV, GenBank accession number is DQ811789, carry out the design of specific primers suitable for ddPCR.

[0045] This example gives the process of screening the best primers, select several pairs of alternative primers designed by software for screening, the alternative primers are as follows, see Table 1.

[0046] Table 1

[0047]

[0048]

[0049] 2. Screening of primers

[0050] (1) The primers of PEDV were randomly matched into four pairs: F1R1, F1R2, F2R1, and F2R2; the primers of TGEV were randomly matched into four pairs: F1R1, F1R2, F2R1, and F2R2.

[0051] (2) Then the two viruses were used for fluorescent quantitative PCR by dye method, PCR system formula: 2Xone-stepSYBRGreenSupermix10μL, upstream p...

Embodiment 2

[0058] Example 2 Optimization of the annealing temperature of the PCR method for detecting porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus on the ddPCR platform.

[0059] 1. Select the combination of primers and probes: PEDV-F1R1P1+TGEV-F1R1P1.

[0060] 2. Then on the ddPCR platform, ddPCR system formula: 2XOne-stepRT-ddPCRSupermix 10 μL, upstream primer, downstream primer 1 μL, probe 0.5 μL, RNAseFreedH 2 O3 μL, positive template 2 μL, total volume 20 μL (concentration of both primers and probes is 10 μM). Do 8 replicate wells and generate microdroplets.

[0061] 3. Amplify on a PCR instrument. The PCR amplification program is reverse transcription at 50°C for 10 minutes; pre-denaturation at 95°C for 10 minutes; denaturation at 94°C for 30 sec, and annealing at 50-60°C (8 temperature gradients are automatically distributed by the instrument) for 60 sec, a total of 40 sec. cycle; 98 ° C for 10 min to end the reaction. Detection on the droplet...

Embodiment 3

[0063] The optimization experiment of embodiment 3 primers, probe concentration

[0064] The concentration of the designed primers and probes was 10 μM, and the combination was PEDV-F1R1P1+TGEV-F1R1P1. The system configuration method is shown in Table 2.

[0065] Table 2

[0066]

[0067]

[0068] Then micro-droplets were generated on the ddPCR platform, transferred to a 96-well plate, and sealed with an aluminum film.

[0069] Amplify on a PCR instrument. The amplification program of PCR is reverse transcription at 50°C for 10 minutes; pre-denaturation at 95°C for 10 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 60 seconds, a total of 40 cycles; and 98°C for 10 minutes to end the reaction. Detection on the droplet digital PCR detector.

[0070] Analysis results: According to the experimental results, the formula of PEDV-F2R2P2+TGEV-F2R2P2 primers and probes was selected, and the selected primers were 1.8 μL and the probes were 0.4 μL.

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Abstract

The invention provides a specific primer and probe combination for detecting porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV). The specific primer and probe combination comprises two pairs of specific primers and two specific probes used in conjunction with the primers. The invention provides a detection kit or a detection reagent for detecting PEDV and TGEV at the same time. The specific primer and the detection kit or the detection reagent provided by the invention have the advantages of rapidness, sensitivity, specificity and the like, and can lay a foundation for double digital PCR (Polymerase Chain Reaction) absolute quantitative detection of PEDV and TEGV, epidemiological investigation, vaccine usage and the like.

Description

technical field [0001] The invention relates to the technical field of detection kits, in particular to a digital PCR absolute quantitative detection method for porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus and a detection kit. Background technique [0002] Porcine epidemic diarrhea (porcineepidemicdiarrhea, PED) is one of the swine diseases occurring worldwide, characterized by diarrhea, vomiting, dehydration and high lethality to suckling piglets. PEDV and porcine transmissible gastroenteritis virus (TGEV) belong to Nidoviridae, Coronaviridae, and Coronaviridae. The pathogenic mechanism and clinical symptoms of the two viruses are very similar, which brings serious harm to the pig industry. . The main source of infection for these two diseases is sick pigs, which spread the virus through feces excreted by sick pigs, and then pollute feed, drinking water and the surrounding environment. Healthy pigs can develop through oral contact with fe...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/686
Inventor 陈晨柴方红王楠于钦磊穆国冬马付坤闫慧张静依
Owner BEIJING SEAGULL BIOVENTURES & BIOTECH CO LTD
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