CN114381554A - CDA primer pair and kit for detecting porcine circovirus type II and application of CDA primer pair and kit - Google Patents
CDA primer pair and kit for detecting porcine circovirus type II and application of CDA primer pair and kit Download PDFInfo
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Abstract
The invention relates to a primer pair based on Closed loop mediated Isothermal Amplification of nucleic acids (CDA) for rapid detection of porcine circovirus type II. The invention discloses a CDA primer pair for detecting porcine circovirus type II, a kit and application thereof. The CDA primer pair is a primer for amplifying a fragment of a conservation region of the porcine circovirus type II and is PCV2-MF/PCV 2-MR; wherein the nucleotide sequence of PCV2-MF is shown as SEQ ID NO.1, and the nucleotide sequence of PCV2-MR is shown as SEQ ID NO. 2. The method provided by the invention can complete the rapid and accurate detection of the porcine circovirus type II without expensive instruments and complex operation, so that the method is suitable for the rapid field detection of customs, farms and the like with low professional degree.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a CDA primer pair for detecting porcine circovirus type II, a kit and application thereof.
Background
The infection of porcine circovirus type II (PCV2) is a type II infectious disease, is a frequently-occurring toxic infectious disease of pigs, and is a new infectious disease of pigs caused by porcine circovirus. The disease can cause serious immunosuppression of swinery, thus easily causing secondary or concurrent infectious diseases. Porcine circovirus type 2 is compromised by the ability to compromise the immune function of the infected pig, and this virus, which causes immunosuppression in the body, is often overlooked due to its frequent occurrence as a subclinical infection. In order to better control the infection of porcine circovirus, it is urgently needed to find a suitable rapid and accurate diagnosis method.
At present, the immunological diagnostic method of PCV2 is less researched, and the early epidemic prevention and control of PCV2 are difficult to apply. In the molecular biology detection technology, the classical PCR technology is widely applied to the detection of various pathogens. The existing detection methods mainly comprise PCR, DNA probe technology and the like. Although these techniques allow rapid detection of the amount of total or pathogenic porcine circovirus type II in a sample, these methods rely on specialized and expensive equipment and are complicated and time consuming to perform (including DNA isolation, hybridization, colorimetry, and probe and primer synthesis), and only those skilled in the art will be able to perform the tests. Therefore, the above methods for nucleic acid molecules are not suitable for rapid detection in situ, and the establishment of a rapid, sensitive and specific method for detecting porcine circovirus type II nucleic acid is imminent.
A Closed loop mediated Isothermal Amplification technology (CDA) of nucleic acid is a method for replacing Japanese LAMP nucleic acid Amplification developed by national Ningbo life and health industry research institute (Chinese patent No. ZL 202110473121.8). The method mainly utilizes 2 different specific primers to identify a specific region of a target gene and carries out amplification reaction under a constant temperature condition. Compared with the conventional gene detection means (such as PCR and the like), the CDA reaction can be finished in a constant-temperature water bath box, the requirement on instruments and equipment is low, the operation is simpler than that of the traditional PCR and culture method, the CDA reaction can be finished accurately without professional personnel, and the amplification result can be combined with the metal ion indicator Hydroxy Naphthol Blue (HNB), so that the result can be judged by observing the color change by naked eyes, and the CDA reaction is suitable for basic medical institutions and local inspection and quarantine departments. In addition, the CDA can greatly shorten the operation time and reduce the sample pollution chance, and is suitable for the rapid diagnosis of porcine circovirus type II. In addition, the key loop forming primer used by the CDA is about 30bp, which is shorter than the loop forming primer in LAMP by 40bp, so that the cost is greatly saved.
Disclosure of Invention
The invention aims to provide a CDA primer pair for detecting porcine circovirus type II, a kit and application thereof.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a CDA primer pair for detecting porcine circovirus type II for non-disease diagnosis purposes is PCV2-MF/PCV2-MR, wherein the nucleotide sequence of PCV2-MF is shown as SEQ ID NO.1, and the nucleotide sequence of PCV2-MR is shown as SEQ ID NO. 2.
The invention also provides a kit for detecting porcine circovirus type II, and the kit comprises the CDA primer pair.
In a preferred embodiment, in the CDA primer pair, the concentration of the primers PCV2-MF and PCV2-MR is 1-2 mu M.
As a preferred embodiment, the kit further comprises Bst polymerase, CDA reaction buffer solution, ultrapure water and color developing agent.
In a preferred embodiment, the color-developing agent is one selected from SYBR green I, Eva green, hydroxynaphthol blue and chrome black T.
As a preferred embodiment, the CDA reaction buffer comprises Tris-HCl, KCl, (NH)4)2SO4、MgSO4And Triton X-100.
As a preferred embodiment, the reaction system of the kit comprises the following components:
2-50mM Tris-HCl pH8.8
2-20mM KCl
2-20mM(NH4)2SO4
2～20mM MgSO4
0.1～0.5％TritonX-100
0.2-1M betaine
1～1.6mM dNTP
5-10U Bst DNA polymerase
100-150 mu mol/L color developing agent
1-2 mu M primer PCV2-MF
1-2 mu M primer PCV 2-MR;
the reaction solvent is ultrapure water.
The invention also provides a method for detecting the porcine circovirus type II by using the kit for non-disease diagnosis, which comprises the following steps:
and 2, reacting the prepared amplification reaction liquid at 60-65 ℃ for 20-80 min, and judging whether the sample contains porcine circovirus type II according to a color development result.
The invention also provides application of the CDA primer group in non-disease diagnosis porcine circovirus type II detection.
The invention also provides application of the kit in porcine circovirus type II detection for non-disease diagnosis.
Compared with the prior art, the invention has the beneficial effects that:
1. the CDA primer pair provided by the invention is obtained by designing and screening a specific conserved region (GenBank: MT814851.1) of the porcine circovirus type II, and the effect of using the outer primer is not obvious in the screening process, so that the CDA primer pair can meet the detection requirement only by consisting of 2 inner primers, namely the inner primer pair PCV2-MF/PCV 2-MR. The primer group provided by the invention has high sensitivity and strong specificity, and the kit prepared from the primer group can quickly and accurately detect the porcine circovirus type II contained in a sample to be detected. The nucleotide sequences of the specific primers are shown in the following table 1:
TABLE 1 primer sequences
| Serial number | Primer name | Nucleotide sequence |
| SEQ ID NO.1 | PCV2-MF | CCGGGTCTGCACAGAAGCGTGATTGGAA |
| SEQ ID NO.2 | PCV2-MR | AAACCACATACGGTAACCATCCCACCAC |
2. The primer pair provided by the invention has extremely high specificity, so that the time required by CDA amplification is short, the detection time is further shortened, and the operation is simplified.
3. The method or the kit provided by the invention can complete the rapid and accurate detection of the porcine circovirus type II without expensive instruments and complex operation, so that the method or the kit is suitable for the rapid field detection of airports, customs, ports, farms and the like with low professional degree. The visual kit provided by the invention provides great convenience for field detection and can realize quick and accurate detection of porcine circovirus type II.
Drawings
FIG. 1 is a graph showing the change of fluorescence intensity with reaction time in example 1 of the present invention.
FIG. 2 is a graph showing the change of fluorescence intensity with reaction time in example 2 of the present invention.
Fig. 3 is a terminal monitoring diagram based on color change for reaction with HNB in embodiment 3 of the present invention.
Detailed Description
The technical solution of the present invention will be described in detail with reference to examples. The experimental procedures used in the following examples are, unless otherwise specified, conventional and may be carried out in accordance with the procedures specified in molecular cloning, a laboratory manual (third edition) J. SammBruke, or in accordance with kits and product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 verification of amplification reaction on porcine circovirus type II conserved sequence DNA fragment by Eva Green
Similar to SYBR Green I, Eva Green is a dye which is combined with all DNA double helix minor groove regions and has Green excitation wavelength, and the inhibition effect on nucleic acid amplification reactions such as PCR and the like is far smaller than that of the dye. In the free state, Eva Green emits weak fluorescence, but once bound to double-stranded DNA, fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of Eva Green is related to the amount of double-stranded DNA, and the amount of double-stranded DNA existing in the nucleic acid amplification system can be detected according to the intensity of the fluorescence signal.
The reaction solutions were combined as follows (the remainder was added ddH)2O to 25 μ L):
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1×Eva Green(Biotum)
Primer:
1600nM PCV2-MF (SEQ ID NO. 1)
1600nM PCV2-MR (SEQ ID NO. 2)
Target is porcine circovirus II type conserved sequence DNA fragment (shown in SEQ ID NO. 3)
A control group without target was also set.
The constant SLAN 96real time PCR reaction temperature is set to 63 ℃, and the reaction time is set to 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 1. The results in FIG. 1 show that: the primer group provided by the invention can realize rapid amplification of the porcine circovirus type II specific conserved region, the fluorescent detection is applied to the primer group, the purpose of real-time monitoring can be realized, and the result can be judged in advance by observing a real-time amplification curve.
Example 2 verification of amplification reaction for porcine circovirus type II extracted genome Using Eva Green
The procedure is as in example 1.
The reaction solutions were combined as follows (the remainder was added ddH)2O to 25 μ L):
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1×Eva Green(Biotum)
Primer:
1600nM PCV2-MF (SEQ ID NO. 1)
1600nM PCV2-MR (SEQ ID NO. 2)
The target is porcine circovirus II extracted genome, and a control group without the target is arranged at the same time.
The constant SLAN 96real time PCR reaction temperature is set to 63 ℃, and the reaction time is set to 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 2. The results in FIG. 2 show that: the primer group provided by the invention can realize rapid amplification of porcine circovirus II extracted genome, the purpose of real-time monitoring can be realized by applying fluorescence detection to the primer group, and the result can be judged in advance by observing a real-time amplification curve.
Example 3 application of Hydroxynaphthol blue (HNB) to endpoint monitoring of amplification reaction of porcine circovirus type II CDA
Hydroxynaphthol blue (HNB) belongs to a metal ion indicator, and aims at the change of the amount of magnesium ions or manganese ions combined with a byproduct pyrophosphate in the reaction, so that different indicating colors are presented to judge the result.
The combination of reaction solutions for amplification of porcine circovirus type II CDA using hydroxynaphthol blue (HNB) is shown below.
The reaction solutions were combined as follows, and ddH was added to the rest2O to 25. mu.L
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
120μM HNB
Primer:
1600nM PCV2-MF (SEQ ID NO. 1)
1600nM PCV2-MR (SEQ ID NO. 2)
The target nucleic acid is porcine circovirus II extracted genome DNA. The amplification reaction was set up with 8 positive controls and 8 negative controls. The reaction temperature of the water bath kettle is set to be 63 ℃ constantly, and the reaction time is 60 min. The end-point results of the negative-positive reactions are shown in FIG. 3, where violet indicates negative and sky blue indicates positive. The experimental results show that: the HNB is applied to the reaction product, so that the reaction result can be judged by observing color change through naked eyes.
The above description is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the spirit of the invention, and any changes and modifications made are within the scope of the invention.
Sequence listing
<110> national institute of Ningbo Life and health industry
NINGBO HUAMEI HOSPITAL University OF CHINESE ACADEMY OF SCIENCES
<120> CDA primer pair and kit for detecting porcine circovirus type II and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213> primer (primer)
<400> 1
ccgggtctgc acagaagcgt gattggaa 28
<210> 2
<211> 28
<212> DNA
<213> primer (primer)
<400> 2
aaaccacata cggtaaccat cccaccac 28
<210> 3
<211> 322
<212> DNA
<213> primer (primer)
<400> 3
ttccgcgggc tggctgaact tttgaaagtg agcgggaaaa tgcagaagcg tgattggaag 60
acgaatgtac acgtcattgt ggggccacct gggtgtggca aaagcaaatg ggctgctaat 120
tttgcagacc cggaaaccac atactggaaa ccacctagaa acaagtggtg ggatggttac 180
catggtgaag aagtggttgt tattgatgac ttttatggct ggctgccgtg ggatgatcta 240
ctgagactct gtgatcgata tcctttgact gttgagacta aaggtggaac tgtacctttt 300
ttggcccgca gtattctgat ta 322
Claims (10)
1. A CDA primer pair for detecting porcine circovirus type II for non-disease diagnosis purposes is characterized in that: the CDA primer pair is PCV2-MF/PCV2-MR, wherein the nucleotide sequence of PCV2-MF is shown as SEQ ID NO.1, and the nucleotide sequence of PCV2-MR is shown as SEQ ID NO. 2.
2. A kit for detecting porcine circovirus type II is characterized in that: the kit comprises the pair of CDA primers of claim 1.
3. The kit for detecting porcine circovirus type II of claim 2, wherein: in the CDA primer pair, the concentration of primers PCV2-MF and PCV2-MR is 1-2 mu M.
4. The kit for detecting porcine circovirus type II of claim 2, wherein: the kit also comprises Bst polymerase, CDA reaction buffer solution, ultrapure water and color developing agent.
5. The kit for detecting porcine circovirus type II of claim 4, wherein: the color developing agent is selected from one of SYBR green I, Eva green, hydroxyl naphthol blue and chrome black T.
6. The kit for detecting porcine circovirus type II of claim 4, wherein: the CDA reaction buffer comprises: Tris-HCl, KCl, (NH)4)2SO4、MgSO4And Triton X-100.
7. The kit for detecting porcine circovirus type II of claim 2, wherein: the reaction system of the kit comprises the following components:
2-50mM Tris-HCl pH8.8
2-20mM KCl
2-20mM(NH4)2SO4
2～20mM MgSO4
0.1～0.5％TritonX-100
0.2-1M betaine
1～1.6mM dNTP
5-10U Bst DNA polymerase
100-150 mu mol/L color developing agent
1-2 mu M primer PCV2-MF
1-2 mu M primer PCV 2-MR;
the reaction solvent is ultrapure water.
8. The method for detecting porcine circovirus type II, for non-disease diagnostic purposes, of the kit of claim 7, comprising the steps of:
step 1: mixing a nucleic acid sample to be detected with a reaction system of the kit to prepare an amplification reaction solution;
step 2: and (3) reacting the prepared amplification reaction solution at 60-65 ℃ for 20-80 min, and judging whether the sample contains porcine circovirus type II according to a color development result.
9. Use of the CDA primer pair of claim 1 in porcine circovirus type II detection for non-disease diagnostic purposes.
10. Use of a kit according to any one of claims 2 to 7 for the detection of porcine circovirus type II for non-disease diagnostic purposes.
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