CN111733294A - Identification primer, identification method and kit for type 4 of Borna psittaci virus - Google Patents
Identification primer, identification method and kit for type 4 of Borna psittaci virus Download PDFInfo
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Abstract
The invention provides an identification primer, an identification method and a kit for a type 4 Bahner parrot virus, and relates to the field of biological detection and disease diagnosis. The sequence of the fluorescent quantitative PCR identification primer is shown as SEQ ID No.1 and SEQ ID No. 2. The identification method of the invention comprises the following steps: s1, extracting the virus RNA of the psittacosis pathogenic tissue, and reversely transcribing the virus RNA into cDNA; s2, taking the cDNA in the step S1 as a template, and carrying out SYBR Green I fluorescent quantitative PCR amplification by using the identifying primer; and S3, analyzing the amplification curve, judging as positive PaBV-4 when the ct value is less than or equal to 30 and the amplification curve is S-shaped, and judging as negative PaBV-4 when the ct value is more than 30. The primer and the identification method have the characteristics of high sensitivity, strong specificity and good repeatability, can be used for detecting the type 4 of the psittacosis Bohr Na virus, and are particularly suitable for detecting and identifying the early-stage and low-content recessive infection viruses of the psittacosis Bohr Na 4 virus.
Description
Technical Field
The invention relates to the field of biological detection and disease diagnosis, in particular to an identification primer, an identification method and a kit for a type 4 Bauerna parrot virus.
Background
The psittacosis borna virus (Parrot borne, PaBV) is a nonsegmented single negative strand RNA virus with a genome size of about 8900bp, which replicates and proliferates in the nucleus with a capsular coat. In the last 70 th century, an atrophy disease, known as hypogastric dilation (PDD), was reported in macarots. The parrot with PDD has the clinical manifestations of severe vomiting, appetite reduction, depression and disordered feather, and in severe cases, nervous symptoms such as ataxia, blindness or tremor can appear. Invasion of the digestive nervous system by viral infection leads to gastrointestinal dysfunction, symptoms of diarrhea and inability to digest food normally, undigested feed in the feces and sometimes even hematochezia. Diseased parrots slowly lose wasting and failing due to vomiting and gastrointestinal dysfunction, and eventually die often due to hunger or secondary bacterial infection, and death of parrots usually occurs within 15 days after the first clinical symptoms appear; some parrots can recover health within a few days after showing obvious clinical symptoms without repeated attacks.
Until 2008, the pathogenic factor of PDD was determined to be a psittacosis Borna virus, 8 genotypes of PaBV have been reported at present, all PaBV can infect parrots to cause PDD, and the discovered virus tropism range is wider, and pathological changes are more complex. Of the 8 genotypes of the psittacosis borna virus, genotype 4 (PaBV-4) is widespread worldwide. In recent years, parrot infection PaBV-4 sometimes happens in China to cause death, but related research reports are few in China. Therefore, if a rapid, specific and sensitive PaBV-4 detection method can be established, the method has important significance for effectively preventing and controlling the prevalence of the Borna virus and promoting the healthy development of parrot culture.
Disclosure of Invention
Therefore, it is necessary to provide a fluorescent quantitative PCR identification primer for the type 4 of the psittacosis Borna virus, which is designed according to the high-conservative region M gene of PaBV-4 and can efficiently, quickly and accurately identify the PaBV-4 virus.
A fluorescent quantitative PCR identification primer for the type 4 of the parrot Borna virus, wherein the primer sequence is as follows:
PaBV4M-F2：GGAGATAGACTTTGTTGGTG(SEQ ID No.1)，
PaBV4M-R2：TCGTGTATTTGGAAATAGACG(SEQ ID No.2)。
the primer is designed according to the high-conservative region M gene of PaBV-4, and can efficiently, quickly and accurately identify the type 4 of the psittacosis Borna virus.
Specifically, the M gene sequence (JX065209.1) of PaBV-4 was first obtained from NCBI database based on the viral matrix protein gene (M gene) as the target gene, and the M gene amplification primers were designed using oligo7.0 software, and the primer sequences were as follows:
PaBV4M-F1：TGAATTCAAAACACACCTACGT(SEQ ID No.3)，
PaBV4M-R1：TTAAGGGCCGGAATGGC(SEQ ID No.4)。
a PaBV-4cDNA is taken as a template, PaBV4M-F1(SEQ ID No.3) and PaBV4M-R1(SEQ ID No.4) are taken as primers, an M gene fragment is amplified by PCR, the size of the fragment is 429bp, and the gene sequence is as follows:
ATGAATTCAAAACACACCTACGTTGAGCTCAAGGACAAGGTAATTGTTCCTGGATGGCCGACACTGATGCTGGAGATAGACTTTGTTGGTGGGACGTCTCATAACCAATTTATCAACATACCGTTTCTTTCAGTGAAGGAGCCACTTCAGCTTCCAAGGGAGAAGAAGCTTGTGGATTACTTGACAATTGATGTTGAGCCCTCTGGACACTCAACAGTTAACGTCTATTTCCAAATACACGACTTCCTGGTCTTAACTCTCAACTCAATATCTGTATACAAAGATCCGCTCAAACCTTTTATGTTCATTAGACTATCAGAGCAACAAAGCAAACACGCAATCAATGCTGCATTCAACGTATTCTCATTTCGTCTTCGGAACATTGGTGTTGGGCCTTTGGGTCCAGACATACGCCATTCCGGCCCTTAA(SEQ ID No.5)。
the M gene sequence (SEQ ID No.5) is used as a reference sequence, and fluorescence quantitative PCR identification primers PaBV4M-F2(SEQ ID No.1) and PaBV4M-R2(SEQ ID No.2) are designed by utilizing Oligo7.0 software.
The invention also provides a fluorescent quantitative PCR identification method of the type 4 Bahner Narrow virus, which comprises the following steps:
s1, extracting the virus RNA of the psittacosis pathogenic tissue, and reversely transcribing the virus RNA into cDNA;
s2, using the cDNA in the step S1 as a template, and carrying out SYBR Green I fluorescent quantitative PCR amplification by using the primers PaBV4M-F2 and PaBV4M-R2 as described in claim 1;
s3, analyzing the amplification curve, and judging as positive PaBV-4 when the ct value is less than or equal to 30 and the amplification curve is S-shaped; when the ct value is more than 30, the PaBV-4 type is judged to be negative.
The identification method can efficiently, quickly and accurately identify whether the PaBV-4 infection exists in the sample, and can reflect the relative content of the virus, and the method can play an important role in timely detecting the Borna 4 type virus of the parrots such as the blue and yellow adamantine parrots, green wing adamantine parrots and the like which breed rare birds in captivity.
In one embodiment, the cDNA content is greater than or equal to 6.7 × 102Copies/. mu.L.
In one embodiment, in step S1, a cDNA sample is prepared using AxyPrep Body Fluid Viral DNA/RNAminiprep Kit 250-prep RNA extraction Kit and PeimeScript reverse transcription Kit
In one embodiment, the PCR amplification reaction procedure of step S2 is:
pre-denaturation at 95 ℃ for 3min, 30 cycles;
denaturation at 95 ℃ for 30 s;
annealing at 53.7 ℃ for 30 s;
extension at 72 ℃ for 20 s;
extension at 72 ℃ for 10 min.
In one embodiment, the PCR amplification reaction system of step S2 includes the following components: TB GreenPremix Ex Taq II, primers PaBV4M-F2 and PaBV4M-R2, ddH2O, cDNA template.
In one embodiment, the PCR amplification reaction system is 25 μ L, and specifically includes:
the invention also provides a kit for identifying the type 4 of the parrot bordetella virus, which comprises the following components: TBGreen Premix Ex Taq II, PaBV4M-F2, PaBV4M-R2, ddH2O, cDNA template.
The kit can accurately, efficiently and quickly identify whether the PaBV-4 type infection exists in the sample, and the sensitivity is the lowest and can be detected as 6.7 × 102Copy/. mu.L, 100 times higher than the normal PCR assay.
The invention also provides application of the kit in identifying the genotype of the Bohr's Nautilus virus.
Compared with the prior art, the invention has the following beneficial effects:
the primer sequence of the invention is designed according to the high-conservative region M gene of PaBV-4, and can efficiently, quickly and accurately identify the type 4 of the psittacosis Borna virus. The fluorescent quantitative PCR identification method can accurately diagnose whether the parrot is infected by the virus, is particularly suitable for detecting and identifying early and low-content recessive infection viruses of the parrot Borna type 4 virus, provides a reliable method for clinical diagnosis and molecular epidemiological investigation of the parrot Borna virus, and has important clinical significance for prevention of the genotype Borna virus.
Drawings
FIG. 1 shows the results of real-time fluorescence quantitative PCR sensitive test by SYBR Green I;
wherein, the ratio of 1-6: 6.7 × 106～6.7×101Copy/. mu.L of sample amplification curve; 7: and (5) negative control.
FIG. 2 shows the results of a general PCR sensitivity test;
wherein M is DNA molecular mass standard and 1-7: 6.7 × 109～6.7×103Copies/. mu.L of sample; 8: and (5) negative control.
FIG. 3 is a SYBR Green I real-time fluorescent quantitative PCR specific amplification curve;
wherein, the ratio is 1: 6.7 × 104Copies/. mu.L of sample; 2: H5N6 subtype avian influenza cDNA; 3: H7N9 subtype avian influenza cDNA; 4: H9N2 subtype avian influenza cDNA; 5: NDV newcastle disease virus cDNA; 6: infectious bronchitis virus cdna (ibdv); 7: and (5) negative control.
FIG. 4 is a clinical sample detection amplification curve;
wherein, 1-8: parrot clinical samples; 9: and (5) negative control.
FIG. 5 shows the results of the general RT-PCR assay;
wherein, 1-8 parts of parrot clinical samples; 9: and (5) negative control.
Detailed Description
To facilitate an understanding of the invention, a more complete description of the invention will be given below in terms of preferred embodiments. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the following examples, the AxyPrep Body Fluid Viral DNA/RNAminiprep Kit 250-preprNA extraction Kit is a product of corning Life sciences (Wu Jiang) Co., Ltd; PeimeScript reverse transcription Kit, pMD19-T vector, DH5 alpha competent cell, TaKaRaMiniBEST Plasmid Purification Kit Ver.4.0 Plasmid extraction Kit, TB Green Premix Ex Taq II TaKaRa company products; DreamTaq Green PCR Master Mix (2X) is a product of Thermo Scientific; the DNA Marker is a product of Guangzhou Dongsheng Biotechnology limited company.
Example 1
SYBR Green I real-time fluorescent quantitative PCR sensitive test.
1. Gradient dilution of known positive sample by 10-fold gradient dilution to 6.7 × 106～6.7×101Copies/. mu.L of 6 serial dilutions of positive samples.
SYBR Green I real-time fluorescent quantitative PCR detection:
the real-time fluorescent quantitative PCR detection takes 6 sample cDNA in 1 as a template, takes PaBV4M-F2 and PaBV4M-R2 as primers and adopts TB Green Premix Ex Taq II (a product of TaKaRa company); DreamTaq Green PCR Master Mix (2 ×) (product of Thermo Scientific Co.) was subjected to PCR reaction, and amplification was performed on a CFX96 real-time fluorescent quantitative PCR instrument (Bio-Rad Co.).
The amplification system is as follows: the total volume was 25. mu.L, as follows: TB Green Premix Ex Taq II 12.5 mu L, PaBV4M-F21 mu L, PaBV4M-R21 mu L, ddH2O9.5 mu L, cDNA template 1 mu L, and ddH for negative control cDNA2O is replaced, and the other reagents are unchanged.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3min, 30 cycles: denaturation at 95 ℃ for 30 s; annealing at 53.7 ℃ for 30 s; extension at 72 ℃ for 20 s; extension at 72 ℃ for 10 min.
3. General PCR sensitivity test
The 6 sample cDNAs in 1 were used as templates for ordinary PCR detection. Reaction system: premix Ex Taq 12.5. mu.L; 1 mu L of each of PaBV4M-F2 and PaBV 4M-R2; 1 mu L of template; ddH2O was supplemented to 25. mu.L. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; 30 cycles: denaturation at 95 ℃ for 30s, annealing at 63-48 ℃ for 30s, and extension at 72 ℃ for 20 s; extension at 72 ℃ for 10 min. The PCR product was detected by electrophoresis on a 1% agarose gel.
4. And (4) analyzing results:
the results show that the lowest copy number detected by the fluorescent quantitative PCR is 6.7 × 102Samples at copy/. mu.L (FIG. 1), whereas the lowest copy number of 6.7 × 10 could be detected by ordinary PCR4Copies/. mu.L of sample (FIG. 2). Therefore, the sensitivity of the established SYBRGreen I real-time fluorescent quantitative PCR method is 100 times higher than that of the common method.
Example 2
SYBR Green I real-time fluorescent quantitative PCR specificity test.
1. Sample preparation avian influenza cDNA (including H5N6, H7N9 and H9N2 subtypes), Newcastle disease Virus cDNA (NDV), avian infectious bronchitis Virus cDNA (IBDV) were provided by the laboratory as 6.7 × 10 diluted in example 14The sensitivity test was carried out using the copy/. mu.L sample as a test sample.
2. The real-time fluorescent quantitative PCR detection is carried out by using cDNA of H5N6, H7N9, H9N2, NDV and IBDV in 1 and 6.7 × 104Copy/. mu.L sample is used as template, PaBV4M-F2 and PaBV4M-R2 are used as primers, TB Green Premix Ex Taq II (product of TaKaRa) and DreamTaq Green PCR Master Mix (2 ×) (product of Thermo Scientific) are used for PCR reaction, and amplification is carried out in CFX96 real-time fluorescent quantitative PCR instrument (Bio-Rad). The amplification system is that the total volume is 25. mu.L, specifically, TB Green Premix Ex Taq II 12.5. mu. L, PaBV 4M-F21. mu. L, PaBV 4M-R21. mu. L, ddH2O9.5. mu. L, cDNA template 1. mu.L, ddH for negative control cDNA2O is replaced, and the other reagents are unchanged.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3min, 30 cycles: denaturation at 95 ℃ for 30 s; annealing at 53.7 ℃ for 30 s; extension at 72 ℃ for 20 s; extension at 72 ℃ for 10 min.
3. Results analysis H5N6, H7N9, H9N2, NDV, IBDV and negative control all had no amplification curve, and showed a curve of 6.7 × 104The copy/. mu.L sample exhibited a standard sigmoidal curve with a ct value of about 22 (FIG. 3). The test result shows that the fluorescence quantitative detection method has good specificity.
Example 3
Sample detection and validation experiments.
1. Collecting pathological tissues: glandular stomach, brain and other tissues 8 parts are aseptically collected from cases of dead parrots with suspected clinical symptoms housed in different zoos.
2. Pathological tissue treatment
Adding 5ml of PBS solution, grinding the pathological tissue by a handheld high-speed homogenizer (Jingxin technology), and taking 1.5ml of supernatant for later use.
3. Viral RNA extraction and reverse transcription
The AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit 250-prep RNA extraction Kit is adopted to complete the extraction of Viral RNA according to the extraction method and the steps of the Kit, and the PeimeScript reverse transcription Kit is adopted to carry out the reverse transcription of the virus to prepare Viral cDNA for later use.
4. Primer design and Synthesis
An M gene fragment 428bp (SEQ ID No.5) is used as a reference sequence, and Oligo7.0 software is utilized to design fluorescent quantitative PCR primers PaBV4M-F2 and PaBV 4M-R2.
SYBR Green I real-time fluorescent quantitative PCR detection
The real-time fluorescent quantitative PCR detection takes cDNA in 3 as a template, takes PaBV4M-F2 and PaBV4M-R2 as primers and adopts TB Green Premix Ex Taq II (a product of TaKaRa company); DreamTaq Green PCR Master Mix (2 ×) (product of Thermo Scientific Co.) was subjected to PCR reaction, and amplification was performed on a CFX96 real-time fluorescent quantitative PCR instrument (Bio-Rad Co.).
The amplification system is as follows: the total volume was 25. mu.L, as follows: TB Green Premix Ex Taq II 12.5 mu L, PaBV4M-F21 mu L, PaBV4M-R21 mu L, ddH2O9.5 mu L, cDNA template 1 mu L, and ddH for negative control cDNA2O is replaced, and the other reagents are unchanged.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3min, 30 cycles: denaturation at 95 ℃ for 30 s; annealing at 53.7 ℃ for 30 s; extension at 72 ℃ for 20 s; extension at 72 ℃ for 10 min.
6. Analysis of results
The result of detecting 8 parrot clinical disease material samples shows that the PaBV-4 type virus is infected by 6 positive parts, and the positive detection rate is 75 percent. As shown in FIG. 4, the No. 1-6 amplification curves ct is less than or equal to 30 and S-shaped, and the PaBV-4 is judged to be positive; the No. 7-8 amplification curve has a ct value larger than 30, and is judged to be negative by PaBV-4; among them, No. 9 is a negative control.
7. Contrast verification
And (3) detecting the 8 parrot clinical disease samples by using an RT-PCR method, and comparing and verifying the results with the results in the step 6. The RT-PCR method comprises the following specific steps:
a PaBV-4 identifying primer was synthesized, with the following sequences, and the amplified fragment was predicted to be about 350 bp:
F3：CAAGGTAATYGTYCCTGGATGG(SEQ ID No.6)，
R3：ACCAATGTTCCGAAGMCGAWAY(SEQ ID No.7)。
respectively taking 8 cDNA samples in the step 6 as templates to carry out PCR reaction, wherein the reaction system comprises the following steps: premix Ex Taq 12.5. mu.L; f3 and R3 are respectively 1 mu L; 1 mu L of template; ddH2O was supplemented to 25. mu.L. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; 30 cycles: denaturation at 95 ℃ for 30s, 5 annealing at 53 ℃ for 30s, and extension at 72 ℃ for 20 s; extension at 72 ℃ for 10 min. The PCR products were subjected to 1% agarose gel electrophoresis (see FIG. 5).
From FIG. 5, it was confirmed that the samples 1, 2, 3 and 4 were positive, the samples 5 and 6 were weakly positive, the samples 7 and 8 were negative, and the sample 9 was a negative control. Wherein, after sequencing the amplification products of samples No.1 and No.2 to Rui Boxing science biotechnology limited, the gene bank is logged for Blast analysis, and the similarity with the gene of PaBV-4 in the database is higher than 98%.
From the results, the method disclosed by the invention is matched with the RT-PCR method, and the method disclosed by the invention is high in identification result accuracy. Moreover, by adopting the fluorescent quantitative PCR method, samples 5 and 6 can be directly judged as positive samples, so that the fluorescent quantitative PCR detection method is more sensitive and accurate.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Guangzhou zoo
Identification primer, identification method and kit for <120> Bohr's Najacobra virus type 4
<160>7
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
<210>2
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
tcgtgtattt ggaaatagac g 21
<210>3
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
tgaattcaaa acacacctac gt 22
<210>4
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
ttaagggccg gaatggc 17
<210>5
<211>429
<212>DNA
<213>Borna disease virus
<400>5
atgaattcaa aacacaccta cgttgagctc aaggacaagg taattgttcc tggatggccg 60
acactgatgc tggagataga ctttgttggt gggacgtctc ataaccaatt tatcaacata 120
ccgtttcttt cagtgaagga gccacttcag cttccaaggg agaagaagct tgtggattac 180
ttgacaattg atgttgagcc ctctggacac tcaacagtta acgtctattt ccaaatacac 240
gacttcctgg tcttaactct caactcaata tctgtataca aagatccgct caaacctttt 300
atgttcatta gactatcaga gcaacaaagc aaacacgcaa tcaatgctgc attcaacgta 360
ttctcatttc gtcttcggaa cattggtgtt gggcctttgg gtccagacat acgccattcc 420
ggcccttaa 429
<210>6
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
caaggtaaty gtycctggat gg 22
<210>7
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
accaatgttc cgaagmcgaw ay 22
Claims (9)
1. The fluorescent quantitative PCR identification primer for the type 4 of the parrot Borna virus is characterized by comprising the following primer sequences:
PaBV4M-F2：GGAGATAGACTTTGTTGGTG(SEQ ID No.1)，
PaBV4M-R2：TCGTGTATTTGGAAATAGACG(SEQ ID No.2)。
2. a fluorescent quantitative PCR identification method for the type 4 of the parrot Borna virus is characterized by comprising the following steps:
s1, extracting the virus RNA of the psittacosis pathogenic tissue, and reversely transcribing the virus RNA into cDNA;
s2, using the cDNA in the step S1 as a template, and carrying out SYBR Green I fluorescent quantitative PCR amplification by using the primers PaBV4M-F2 and PaBV4M-R2 as described in claim 1;
s3, analyzing the amplification curve, and judging as positive PaBV-4 when the ct value is less than or equal to 30 and the amplification curve is S-shaped; when the ct value is more than 30, the PaBV-4 type is judged to be negative.
3. The fluorescent quantitative PCR identification method of claim 2, wherein the cDNA content is not less than 6.7 × 102Copies/. mu.L.
4. The method of claim 2, wherein in step S1, cDNA samples are prepared using AxyPrep body fluid Viral DNA/RNA Miniprep Kit 250-prep RNA extraction Kit and PeimeScript reverse transcription Kit.
5. The method for fluorescent quantitative PCR identification as claimed in claim 2, wherein the PCR amplification reaction procedure of step S2 is as follows:
pre-denaturation at 95 ℃ for 3min, 30 cycles;
denaturation at 95 ℃ for 30 s;
annealing at 53.7 ℃ for 30 s;
extension at 72 ℃ for 20 s;
extension at 72 ℃ for 10 min.
6. The fluorescent quantitative PCR identification method of claim 2 or 5, wherein the amplification reaction system of step S2 comprises the following components: TB Green Premix Ex Taq II, primers PaBV4M-F2 and PaBV4M-R2, ddH2O, cDNA template.
7. The method for fluorescent quantitative PCR identification as claimed in claim 6, wherein the PCR amplification reaction system is 25 μ L, and specifically comprises:
8. a kit for identifying type 4 of birnavirus parrot, which comprises the following components: TB GreenPremix Ex Taq II, PaBV4M-F2, PaBV4M-R2, ddH2O, cDNA template.
9. Use of a kit according to claim 8 for the identification of the borna psittaci virus genotype.
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Cited By (1)
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| CN118028544A (en) * | 2024-04-11 | 2024-05-14 | 广东华南珍稀野生动物物种保护中心 | Primer pair for detecting 4-type parrot borna virus N gene and application thereof |
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| CN118028544A (en) * | 2024-04-11 | 2024-05-14 | 广东华南珍稀野生动物物种保护中心 | Primer pair for detecting 4-type parrot borna virus N gene and application thereof |
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Application publication date: 20201002 |