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CDA primer pair and kit for detecting porcine circovirus type II and application of CDA primer pair and kit

The technology of a porcine circovirus and kit is applied in the direction of recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., which can solve the problems of complicated and time-consuming operation process, and achieve shortened detection time, high sensitivity, and short time effect

Pending Publication Date: 2022-04-22
国科宁波生命与健康产业研究院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these techniques can quickly detect the total or pathogenic porcine circovirus type II quantity in the sample, these methods rely on special and expensive equipment, and its operation process is complicated and time-consuming (comprising DNA separation, hybridization, colorimetry, etc.) and the synthesis of probes and primers, etc.), only skilled technicians can complete the test
Therefore, the above methods for nucleic acid molecules are not suitable for on-site rapid detection, and it is imminent to establish a fast, sensitive and specific porcine circovirus type II nucleic acid detection method

Method used

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  • CDA primer pair and kit for detecting porcine circovirus type II and application of CDA primer pair and kit
  • CDA primer pair and kit for detecting porcine circovirus type II and application of CDA primer pair and kit
  • CDA primer pair and kit for detecting porcine circovirus type II and application of CDA primer pair and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Application of Eva Green to Verify the Amplification Reaction of Porcine Circovirus Type II Conserved Sequence DNA Fragment

[0042] Similar to SYBR Green I, Eva Green is a dye with a green excitation wavelength that binds to the minor groove of all DNA double helices, and its inhibitory effect on nucleic acid amplification reactions such as PCR is much smaller than that of the latter. In the free state, EvaGreen emits weak fluorescence, but once bound to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of Eva Green is related to the quantity of double-stranded DNA, and the quantity of double-stranded DNA existing in the nucleic acid amplification system can be detected according to the intensity of the fluorescence signal.

[0043] The reaction solution combination is as follows (all the other add ddH 2 0 to 25 μL):

[0044] 20mM Tris-HCl pH8.8

[0045] 10mM KCl

[0046] 10mM (NH 4 ) 2 SO 4

[004...

Embodiment 2

[0059] Example 2 Application of Eva Green to Verify the Amplification Reaction to Porcine Circovirus Type II Extracted Genome

[0060] Method is with embodiment 1.

[0061] The reaction solution combination is as follows (all the other add ddH 2 0 to 25 μL):

[0062] 20mM Tris-HCl pH8.8

[0063] 10mM KCl

[0064] 10mM(NH4) 2 SO 4

[0065] 14mM MgSO 4

[0066] 0.1% Triton X-100

[0067] 1M betaine

[0068] 1.25mM dNTPs

[0069] 8U Bst DNA polymerase (NEW ENGLAND Biolabs)

[0070] 1 x Eva Green (Biotum)

[0071] Primers:

[0072] 1600nM PCV2-MF (shown in SEQ ID NO.1)

[0073] 1600nM PCV2-MR (shown in SEQ ID NO.2)

[0074] Target: the genome of porcine circovirus type II was extracted, and a control group without target was set at the same time.

[0075] Set the SLAN 96 real time PCR reaction temperature to be constant at 63°C, and the reaction time to be 60min. The curve of fluorescence intensity changing with reaction time is as follows: figure 2 shown. fig...

Embodiment 3

[0076] Embodiment 3 uses hydroxynaphthol blue (HNB) to carry out porcine circovirus type II CDA amplification reaction endpoint monitoring

[0077] Hydroxynaphthol blue (HNB) is a metal ion indicator, which is aimed at the change of the amount of magnesium ions or manganese ions combined with the by-product pyrophosphate in the reaction, so as to present different indicator colors to realize the judgment of the result.

[0078] The composition of the reaction solution for porcine circovirus type II CDA amplification using hydroxynaphthol blue (HNB) is shown below.

[0079] The reaction solution is combined as follows, and ddH is added to the rest 2 0 to 25 μL

[0080] 20mM Tris-HCl pH8.8

[0081] 10mM KCl

[0082] 10mM (NH 4 ) 2 SO 4

[0083] 14mM MgSO 4

[0084] 0.1% Triton X-100

[0085] 1M betaine

[0086] 1.25mM dNTPs

[0087] 8U Bst DNA polymerase (NEW ENGLAND Biolabs)

[0088] 120 μM HNB

[0089] Primers:

[0090] 1600nM PCV2-MF (shown in SEQ ID NO.1)

[...

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Abstract

The invention relates to a method for rapidly detecting a porcine circovirus type II, in particular to a method for rapidly detecting the porcine circovirus type II by using a primer pair based on a closed neck loop-mediated isothermal nucleic acid amplification (CDA) technology. The invention discloses a CDA primer pair for detecting porcine circovirus type II, a kit and application of the CDA primer pair. The CDA primer pair is a primer for amplifying a conserved region fragment of the porcine circovirus type II, and is PCV2-MF / PCV2-MR (porcine circovirus type II); wherein the nucleotide sequence of the PCV2-MF is as shown in SEQ ID NO. 1, and the nucleotide sequence of the PCV2-MR is as shown in SEQ ID NO. 2. According to the method provided by the invention, rapid and accurate detection of the porcine circovirus type II can be completed without expensive instruments or complicated operation, so that the method is suitable for on-site rapid detection of places such as customs and farms with low professional degree.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a pair of CDA primers, a kit and an application thereof for detecting porcine circovirus type II. Background technique [0002] Porcine circovirus type II (PCV2) infection is a second-class infectious disease, a common viral infectious disease of pigs, and a new infectious disease of pigs caused by porcine circovirus. The disease can lead to severe immunosuppression in pigs, which can easily lead to secondary or concurrent other infectious diseases. The danger of porcine circovirus type 2 is that it can damage the immune function of infected pigs. This virus, which can lead to immunosuppression of the body, is often overlooked because it often appears in the form of subclinical infection. In order to better control porcine circovirus infection, it is urgent to find a suitable rapid and accurate diagnostic method. [0003] At present, there are few studi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2531/119C12Q2525/307
Inventor 毛瑞赵月蔡挺
Owner 国科宁波生命与健康产业研究院
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