CN114807440A - Primer and probe for pigeon rotavirus A type fluorescent quantitative PCR detection and kit thereof - Google Patents
Primer and probe for pigeon rotavirus A type fluorescent quantitative PCR detection and kit thereof Download PDFInfo
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Abstract
The invention provides a primer, a probe and a kit for pigeon rotavirus A type fluorescence quantitative PCR detection, wherein the primer sequence is as follows: primer PiRVA-TF: 5'-CCGAACTGCTCCTGTATGAAT-3', primer PiRVA-TR: 5'-CATTGCCCATTGCTATCCATTT-3', respectively; the probe is as follows: probe PiRVA-probe: 5'-AGTCTGACATCCATTTCATTGCACAACC-3', and its 5 '-end marks the fluorescence report group FAM, and marks the quenching group DBQ1 in the middle and 3' -end of the probe, the method of the invention has high sensitivity, good stability, strong specificity, good repeatability, can be used for the detection of pigeon rotavirus A type specificity, and lays the foundation for the follow-up scientific research of pigeon rotavirus A type pathogenesis and the development of molecular epidemiology.
Description
Technical Field
The invention belongs to the field of animal virology, and particularly relates to a primer and a probe for pigeon rotavirus A type fluorescent quantitative PCR detection and a kit thereof.
Background
The pigeon breeding history of human beings is long, the pigeons are domesticated into ornamental pigeons, racing pigeons and meat pigeons, the pigeon breeding house has a strong immune system, and the pigeons are mostly open or semi-open, so that the air circulation is good, the air is fresh, and the pigeons are less sick compared with other livestock and poultry. However, with the rapid development of the large-scale and intensive pigeon breeding industry, the total breeding amount and the breeding density are increased, the breeding mode of the pigeons is changed, and because the breeding management level is low, the consciousness of epidemic disease prevention and treatment is poor, and the trade circulation of the pigeons is frequent (including the competition of homing pigeons), the diseases of the pigeons are more and more serious and more complex. In recent years, pigeon viral infectious diseases seriously threaten the pigeon raising industry, and according to domestic and foreign researches, the reported pigeon viral infectious diseases comprise pigeon Newcastle disease, pigeon rotavirus infection, pigeon adenovirus infection, pigeon circovirus infection, pigeon herpes virus infection, pigeon pox, H9 subtype low-pathogenicity avian influenza and the like.
Rotavirus (Rotavirus, RV) belongs to the genus Rotavirus of reoviridae, and virus particles are non-vesicular membrane, are in a regular icosahedron shape, are divided into 3 layers of shells, have the diameter of about 75nm, and are in a wheel shape under an electron microscope, so the name is obtained. The rotavirus genome consists of 11 segments of double-stranded RNA, and each segment is 663-3302 bp in length. According to the latest classification of the International Committee for the Classification of Viruses on Taxomy of Viruses, ICTV, the genus rotavirus comprises 10 virus species A to J. At present, rotavirus A, D, F and G can infect birds, and at present, rotavirus A, D and G can infect pigeons. The genetic sequences of rotavirus often differ significantly between strains of different hosts of the same virus species. Rotavirus type a (RVA) generally infects a specific population of animals, and such viruses are called homologous strains, but occasionally cross-species transmission is also present. Rotavirus a type hosts are wide and widely present in mammals and birds, and clinical symptoms after infection of birds are mainly manifested by severe diarrhea, dehydration, dysplasia and increased mortality. Pigeon infection with rotavirus a is characterized by vomiting, diarrhea, dehydration and cloaca. The sick pigeons have poor spirit, anorexia and digestive dysfunction at the beginning of illness. Then cachexia, lethargy, anorexia and severe diarrhea, the feces are watery, and are severely dehydrated, emaciated and anemia, and finally die due to exhaustion. The autopsy lesions are mainly manifested by liver necrosis and congestion, hemorrhage and edema of intestinal mucosa, and in addition, the body is dehydrated and cloacal cavities are inflamed. Rotavirus type A infection has seriously affected the healthy development of the pigeon industry.
The Real-time fluorescent quantitative PCR method (Real-time PCR) is a method for detecting the total amount of products after each Polymerase Chain Reaction (PCR) cycle by using fluorescent chemicals in a DNA amplification reaction. Real-time fluorescent quantitative PCR is used for monitoring the PCR process in real time through a fluorescent signal in the PCR amplification process. Since the Ct value of a template is linear with the initial copy number of the template during the exponential phase of PCR amplification. The fluorescent probe method is to use a sequence-specific fluorescent labeled probe to detect a product, and the appearance of the probe method greatly improves the specificity of a quantitative PCR technology compared with the conventional PCR technology. The TaqMan probe method is characterized in that a specific fluorescent probe is added into a Real-time PCR amplification reaction system besides a pair of primers, the probe is only specifically combined with a template, and the combining site of the probe is between the two primers. The 5 'end of the probe is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group. When the probe is complete, the fluorescence excited by the 5 ' end reporter group through the light source of the instrument is just quenched by the near-distance 3 ' end fluorophore, and the instrument cannot detect the fluorescence signal excited by the 5 ' end reporter group. Along with the Real-time PCR, when Taq enzyme meets a probe combined with a template in the chain extension process, the 5 ' -3 ' exonuclease activity (the activity is double-chain specificity, and a free single-chain probe is not influenced) of the Taq enzyme can cut the probe, a 5 ' end reporter group is released to be free in a reaction system, the shielding of a 3 ' end fluorescence quenching group is kept away, and a fluorescence signal emitted by the excited 5 ' end reporter group can be monitored. That is, every time a DNA chain is amplified, a fluorescent molecule is formed, the accumulation of a fluorescent signal and the formation of a Real-time PCR product are completely synchronized, and the intensity of a report signal is related to the copy number of the template DNA. Because a more specific probe is added into the TaqMan real-time fluorescent quantitative probe on the basis of the conventional SYBR Green I real-time fluorescent quantitative PCR method, a positive amplification signal can be detected only by combining a detection target gene with the specificity of the TaqMan probe, so that the detection result is more specific, and the TaqMan real-time fluorescent quantitative probe is widely applied to the field of animal infectious disease detection. In addition, different fluorescence Reporter groups (reporters, R) are marked, so that multiple etiological detections can be simultaneously carried out on multiple samples (especially samples which are difficult to obtain or have low pathogenic load), and the kit has the advantages of high flux, rapid result and the like, and is widely used in the fields of new infectious diseases and the like.
The real-time fluorescent quantitative PCR method of the pigeon rotavirus A type fluorescent probe established by the invention can be used for epidemiological detection, can also be used for accurate quantification of the infection degree of the pigeon rotavirus A type, can be effectively used for research on the difference of a pigeon rotavirus A type pathogenesis, and can fill up the blank of related research fields at home and abroad.
Disclosure of Invention
The invention aims to provide a primer and a probe for pigeon rotavirus A type fluorescence quantitative PCR detection and a kit thereof. The TaqMan probe real-time fluorescence quantitative PCR detection method established by the primer and the probe has the advantages of high sensitivity, good stability, strong specificity and good repeatability, can detect 57 copies/mu L at least, can be used for epidemiological detection, can also be used for accurate quantification of the infection degree of the pigeon rotavirus A, can be effectively used for difference research of the pigeon rotavirus A pathogenic mechanism, and can fill up the blank of related research fields at home and abroad.
The purpose of the invention is realized by the following technical scheme:
a primer and a probe for pigeon rotavirus A type real-time fluorescent quantitative PCR detection are disclosed, wherein the primer sequence is as follows:
primer PiRVA-TF: 5'-CCGAACTGCTCCTGTATGAAT-3' the flow of the air in the air conditioner,
primer PiRVA-TR: 5'-CATTGCCCATTGCTATCCATTT-3', respectively;
the sequence of the probe is as follows:
probe PiRVA-probe: 5'-AGTCTGACATCCATTTCATTGCACAACC-3', and the 5 '-end is marked with a fluorescence reporter group FAM, and the middle and the 3' -end of the probe are respectively marked with a quenching group DBQ 1.
The real-time fluorescent quantitative PCR detection method of the primer and the probe for the pigeon rotavirus A comprises the following steps:
(1) preparation of nucleic acid:
extracting nucleic acid RNA of PiRVA virus according to the instruction method of a nucleic acid extraction Kit (EasyPure Viral DNA/RNA Kit), performing reverse transcription to form cDNA, and storing at-20 ℃ for later use;
(2) construction of Positive Standard
According to the sequence characteristics of the identified pigeon rotavirus A type VP7 gene, a specific primer for PCR amplification is designed, wherein an upstream primer PiRVA-F: 5'-GCCAACCACATCAGTCTACTTA-3', and a downstream primer PiRVA-R: 5'-CACGATGCGACTGTATAATTGA-3', using the prepared PiRVA nucleic acid cDNA as a template, and using an upstream primer PiRVA-F and a downstream primer PiRVA-R to perform PCR amplification, the expected amplified fragment size is 399 bp. Wherein the primer is synthesized by Biotechnology engineering (Shanghai) GmbH.
Amplification was performed using a 50. mu.L system recommended by PCR amplification reagents (2 XPCR Master reagents), in which 25. mu.L of 2 XPCR Master Mix reaction solution, and upstream/downstream primers (PiRVA-F/PiRVA-R) (primer concentration 20. mu. mol. L) -1 ) mu.L of each 1. mu. L, PiRVA nucleic acid cDNA template was supplemented with sterile deionized water to a total reaction volume of 50. mu.L. Mixing, PCR amplification and amplification conditionPre-denaturation at 94 ℃ for 5min, then cycling, denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 35s, and extension at 72 ℃ for 35s, and after 35 cycles, final extension at 72 ℃ for 10 min.
After the PCR reaction is finished, identifying the PCR product by using 1.0% agarose gel electrophoresis, and performing gel cutting and recovery on the specific target fragment by using an agarose gel recovery kit. Cloning the target gene fragment onto a pEASY-T1 vector according to the pEASY-T1 Simple Cloning Kit instruction, randomly picking 5 single colonies, culturing the single colonies in an ampicillin (the content is 100 mu g/mL) resistant LB liquid culture medium for 14h, and then extracting corresponding plasmids by using a rapid plasmid miniextraction Kit. The extracted plasmid is identified by PCR with primers (PiRVA-F/PiRVA-R) and conditions during PCR amplification, and the screened positive recombinant plasmid is sent to the company of Biotechnology engineering (Shanghai) and Limited for sequencing. After Blast analysis, the positive recombinant plasmid that was expected from the experiment was used as the standard (P-PiRVA) in this study. After determination of the concentration by means of a spectrophotometer, the corresponding copy number is calculated to be 5.7X 10 8 Copied/. mu.L, and serially diluted 10-fold to obtain concentrations of 5.7X 10 7 Copies/. mu.L to 5.7X 100 copies/. mu.L, were all frozen at-20 ℃ until use.
(3) Optimizing the real-time fluorescent quantitative PCR reaction condition of the TaqMan probe:
preparing a 20 mu L real-time fluorescent quantitative PCR reaction system according to the specification of a Probe qPCR Mix kit, taking a positive standard substance (P-PiRVA) as a template, and carrying out real-time fluorescent quantitative PCR reaction at different annealing temperatures (56-63 ℃), concentrations (2-20 mu mol/L) of primers (PiRVA-TF and PiRVA-TR) and concentrations (2-20 mu mol/L) of Probe PiRVA-Probe, so as to optimize the reaction conditions.
Optimizing the concentration of the primer: the concentrations of the upstream primer PiRVA-TF and the downstream primer PiRVA-TR are diluted to 2 mu mol/L, 4 mu mol/L, 8 mu mol/L, 10 mu mol/L and 20 mu mol/L respectively for detection, and the optimal concentration of the upstream primer PiRVA-TR and the downstream primer PiRVA-TR is determined to be 10 mu mol/L through analysis and comparison of test results.
Optimizing the concentration of the probe: the concentration of the probe PiRVA-probe is diluted to 2 mu mol/L, 4.0 mu mol/L, 8 mu mol/L, 10 mu mol/L and 20 mu mol/L in a concentration-doubling ratio and then respectively detected, and the optimal concentration of the probe PiRVA-probe is determined to be 10 mu mol/L through analysis and comparison of test results.
When the annealing and extension temperatures are optimized, the selected annealing and extension temperatures are 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃ and 63 ℃, and the optimal annealing and extension temperatures are both 58 ℃ through analysis and comparison of test results.
By the above optimization method: the screened optimal reaction system is as follows: probe qPCR Mix (2X) mixture 10. mu.L, upstream/downstream primers (PiRVA-TF and PiRVA-TR) (10. mu. mol/L) each 0.4. mu.L, Probe (PiRVA-Probe) (10. mu. mol/L) 0.8. mu.L, template 1. mu.L, Water (nucleic-free Water) to make up to 20. mu.L.
The optimized optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 120 s; at 95 ℃ for 10s and at 58 ℃ for 30s, for 40 cycles.
(4) Establishment of TaqMan probe real-time fluorescence quantitative standard curve
Using optimized TaqMan probe to perform real-time fluorescence quantitative PCR reaction conditions with the content of 5.7 multiplied by 10 7 Copy/. mu.L-5.7X 10 2 The amplification was performed using a copy/. mu.L of the standard (P-PiRVA) as a template to obtain an amplification kinetics curve. The common logarithm of the initial copy number of the standard substance is used as an abscissa, a cycle threshold (Ct value) is used as an ordinate, a pigeon rotavirus A type TaqMan probe real-time fluorescence quantitative PCR standard curve (shown in figure 1) is obtained, the slope of the curve is-3.319, the Y-axis intercept is 37.37, the amplification efficiency is 100%, and the correlation coefficient is 1.00, which indicates that the established standard curve of the real-time fluorescence quantitative PCR method has a good linear relation.
Using optimized TaqMan probe to perform real-time fluorescence quantitative PCR reaction conditions respectively with the content of 5.7 multiplied by 10 4 Copies/. mu.L to 5.7X 10 0 The copy/. mu.L standard (P-PiRVA) is used as a template for amplification, and the lowest detection limit of the TaqMan probe real-time fluorescent quantitative PCR method is 57 copies/. mu.L (figure 2).
The invention also provides application of the primers (PiRVA-TF and PiRVA-TR) and the probe (PiRVA-probe) in preparation of a pigeon rotavirus A type specific detection kit.
The invention also provides a pigeon rotavirus A type real-time fluorescent quantitative PCR detection kit, which comprises the primers (PiRVA-TF and PiRVA-TR) and the probe (PiRVA-probe).
Compared with the prior art, the invention has the advantages that:
1. the detection is rapid and efficient: the detection method does not need to carry out conventional agarose gel electrophoresis detection, and the result can be judged by a program carried by a real-time fluorescence quantitative PCR machine after the reaction is finished.
2. The quantification is accurate: through preparing a standard substance and drawing a standard curve, the infection of the pigeon rotavirus A type is directly judged according to the Ct value in a sample to be detected, and the infection degree can be accurately quantified.
3. The sensitivity is high: the lowest detectable 57 copies/. mu.L.
4. The specificity is strong: the fluorescent probe has no reaction signals with common pathogenic pigeon adenovirus type B (PiAdVB), pigeon circovirus (PiCV), pigeon Paramyxovirus (PiNDV), pigeon herpesvirus (PiHV) and H9 subtype avian influenza virus (H9 AIV) in pigeon groups, and only shows fluorescent signals when detecting the rotavirus type A.
5. The repeatability is good: the intra-group variation coefficient of the established real-time fluorescent quantitative PCR detection method for PiRVA detection is 0.27% -1.50%, and the inter-group variation coefficient is 0.24% -1.68%, which shows that the real-time quantitative PCR method established by the invention has good repeatability.
Drawings
FIG. 1 TaqMan probe real-time fluorescent quantitative PCR detection PiRVA standard curve.
FIG. 2 is a diagram showing the results of the TaqMan probe real-time fluorescent quantitative PCR PiRVA sensitivity test. Wherein 1 is template concentration of 5.7 × 10 4 Copy/. mu.L, 2 as template concentration 5.7X 10 3 Copy/. mu.L, 3 as template concentration 5.7X 10 2 Copy/. mu.L, 4 as template concentration 5.7X 10 1 Copy/. mu.L, 5 as template concentration 5.7X 10 0 Copies/. mu.L.
FIG. 3 is a diagram showing the results of the TaqMan probe real-time fluorescent quantitative PCR PiRVA specificity test, wherein 1 is PiRVA; c is a control sample (PiAdVB, PiCV, PiNDV, PiHV, H9 AIV) which is indistinguishable to the naked eye due to the absence of a fluorescent signal.
Detailed Description
The invention is described in detail below with reference to the drawings and examples of the specification:
example 1
1. Relevant test pathogens
The pathogenic pigeon rotavirus A type (PiRVA), the pigeon adenovirus B type (PiAdVB), the pigeon circovirus (PiCV), the pigeon Paramyxovirus (PiNDV), the pigeon herpesvirus (PiHV) and the H9 subtype avian influenza virus (H9 AIV) for the test are identified and stored by the animal husbandry and veterinary institute of agricultural and scientific institute of Fujian province.
2. Preparation of nucleic acids
The nucleic acid DNA (PiCV, PiAdVB, PiHV, without reverse transcription) or the nucleic acid RNA (PiRVA, PiNDV, H9 AIV, reverse transcription into cDNA) of the virus was extracted by using the nucleic acid extraction Kit (Easypure Viral DNA/RNA Kit) of Beijing Quanji Biotech Ltd according to the instruction method, and the extracted nucleic acid DNA or RNA was stored at-20 ℃.
Primer and probe design for TaqMan probe real-time fluorescent quantitative PCR detection method
According to the gene sequences of all pigeon rotavirus A types registered in GenBank, carrying out comparison and analysis of whole genes, and finally selecting specific primers and probes designed by the VP7 gene of the pigeon rotavirus A type, wherein the sequences are as follows:
primer PiRVA-TF (SEQ ID NO. 1): 5'-CCGAACTGCTCCTGTATGAAT-3' the flow of the air in the air conditioner,
primer PiRVA-TR (SEQ ID NO. 2): 5'-CATTGCCCATTGCTATCCATTT-3', respectively;
probe PiRVA-probe (SEQ ID NO. 3):
5’-AGTCTGACATCCATTTCATTGCACAACC-3’
wherein, the probe PiRVA-probe (SEQ ID NO.3) is marked with a fluorescence reporter group FAM at the 5 '-end, and is respectively marked with a quenching group DBQ1 at the middle and the 3' -end, namely the probe is marked with two DBQ1 quenching groups;
both primers and probes were synthesized by Biotechnology engineering (Shanghai) Inc.
4. Construction of Positive Standard
According to the sequence characteristics of the identified pigeon rotavirus A type VP7 gene, a specific primer for PCR amplification is designed, wherein an upstream primer PiRVA-F: 5'-GCCAACCACATCAGTCTACTTA-3', and a downstream primer PiRVA-R: 5'-CACGATGCGACTGTATAATTGA-3', the expected amplified fragment size is 399 bp.
Amplification was performed using a 50. mu.L system recommended by PCR amplification reagents (2 XPCR Master reagents), in which 25. mu.L of 2 XPCR Master Mix reaction solution, and upstream/downstream primers (PiRVA-F/PiRVA-R) (primer concentration 20. mu. mol. L) -1 ) mu.L each, 2. mu.L of template DNA (PiRVA nucleic acid cDNA), and sterile deionized water to a total reaction volume of 50. mu.L. Mixing, performing PCR amplification under the conditions of pre-denaturation at 94 deg.C for 5min, circulating, denaturation at 94 deg.C for 30s, annealing at 54 deg.C for 35s, and extension at 72 deg.C for 35s, and final extension at 72 deg.C for 10min after 35 cycles.
After the PCR reaction is finished, identifying the PCR product by using 1.0% agarose gel electrophoresis, and cutting and recovering the specific target fragment by using an agarose gel recovery kit. Cloning the target gene fragment onto a pEASY-T1 vector according to the pEASY-T1 Simple Cloning Kit instruction, randomly picking 5 single colonies, culturing the single colonies in an ampicillin (the content is 100 mu g/mL) resistant LB liquid culture medium for 14h, and then extracting corresponding plasmids by using a rapid plasmid miniextraction Kit. The extracted plasmid is identified by PCR with primers (PiRVA-F/PiRVA-R) and conditions during PCR amplification, and the screened positive recombinant plasmid is sent to the company of Biotechnology engineering (Shanghai) and Limited for sequencing. After Blast analysis, the positive recombinant plasmid that was expected from the experiment was used as the standard (P-PiRVA) in this study. After determination of the concentration by means of a spectrophotometer, the corresponding copy number is calculated to be 5.7X 10 8 Copied/. mu.L, and serially diluted 10-fold to obtain concentrations of 5.7X 10 7 Copies/. mu.L to 5.7X 100 copies/. mu.L, were all frozen at-20 ℃ until use.
Establishment of 5 pigeon rotavirus A type TaqMan probe real-time fluorescent quantitative PCR specificity detection method
5.1 optimization of real-time fluorescent quantitative PCR reaction conditions by TaqMan probe
Preparing a 20 mu L real-time fluorescent quantitative PCR reaction system according to the specification of a Probe qPCR Mix kit, taking a positive standard substance (P-PiRVA) as a template, and carrying out real-time fluorescent quantitative PCR reaction at different annealing temperatures (56-63 ℃), concentrations (2-20 mu mol/L) of primers (PiRVA-TF and PiRVA-TR) and concentrations (2-20 mu mol/L) of Probe PiRVA-Probe, so as to optimize the reaction conditions.
Optimizing the concentration of the primer: the concentrations of the upstream primer PiRVA-TF and the downstream primer PiRVA-TR are diluted to 2 mu mol/L, 4 mu mol/L, 8 mu mol/L, 10 mu mol/L and 20 mu mol/L respectively for detection, and the optimal concentration of the upstream primer PiRVA-TR and the downstream primer PiRVA-TR is determined to be 10 mu mol/L through analysis and comparison of test results.
Optimizing the concentration of the probe: the concentration of the probe PiRVA-probe is diluted to 2 mu mol/L, 4.0 mu mol/L, 8 mu mol/L, 10 mu mol/L and 20 mu mol/L in a concentration-doubling ratio and then respectively detected, and the optimal concentration of the probe PiRVA-probe is determined to be 10 mu mol/L through analysis and comparison of test results.
When the annealing and extension temperatures are optimized, the selected annealing and extension temperatures are 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃ and 63 ℃, and the optimal annealing and extension temperatures are both 58 ℃ through analysis and comparison of test results.
By the above optimization method: the screened optimal reaction system is as follows: probe qPCR Mix (2X) mixture 10. mu.L, upstream/downstream primers (PiRVA-TF and PiRVA-TR) (10. mu. mol/L) each 0.4. mu.L, Probe (PiRVA-Probe) (10. mu. mol/L) 0.8. mu.L, template 1. mu.L, Water (nucleic-free Water) to make up to 20. mu.L.
The optimized optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 120 s; 40 cycles of 95 ℃ for 10s and 58 ℃ for 30 s.
5.2 establishment of TaqMan Probe real-time fluorescent quantitation Standard Curve
Respectively taking the content of a standard substance (P-PiRVA) as 5.7 multiplied by 10 7 Copies/. mu.L-5.7X 10 2 And (3) taking the copied/mu L standard substance as a template, and carrying out amplification by using the optimized reaction conditions to obtain an amplification kinetic curve. Obtaining real-time fluorescence of a pigeon rotavirus A type TaqMan probe by taking a common logarithm of initial copy number of a standard as an abscissa and a cycle threshold (Ct value) as an ordinateThe quantitative PCR standard curve (see figure 1) has the slope of-3.319, the Y-axis intercept of 37.37, the amplification efficiency of 100 percent and the correlation coefficient of 1.00, and shows that the established standard curve of the real-time fluorescent quantitative PCR method has a good linear relationship.
5.3 TaqMan Probe real-time fluorescent quantitative PCR sensitivity test
Respectively taking the content of a standard substance (P-PiRVA) as 5.7 multiplied by 10 4 Copies/. mu.L to 5.7X 10 0 The copy/. mu.L standard substance is used as a template, and the optimized reaction conditions are used for amplification, so that the lowest detection limit of the obtained TaqMan probe real-time fluorescent quantitative PCR method is 57 copies/. mu.L (figure 2).
5.4 TaqMan Probe real-time fluorescent quantitative PCR specificity test
The established TaqMan probe real-time fluorescent quantitative PCR method is used for respectively detecting PiRVA, PiAdV-B, PiCV, PiNDV, PiHV and H9 AIV. As a result, a positive amplification signal was observed only for PiRVA, and no positive amplification signal was observed for PiAdVB, PiCV, PiNDV, PiHV, H9 AIV (FIG. 3).
5.5 TaqMan Probe real-time fluorescent quantitative PCR repeatability test
The established real-time fluorescent quantitative PCR method is used for respectively measuring the plasmid content to be 5.7 multiplied by 10 7 、5.7×10 5 、 5.7×10 3 The standard of (2) was tested, and the content of each plasmid was repeated 3 times, and intra-group (intra-group) coefficient of variation was calculated. And (3) respectively subpackaging the standard substances with different plasmid contents, storing at-20 ℃, taking out every 7 days, detecting by using the established real-time fluorescence quantitative PCR method for 3 times, and calculating the inter-group (inter-group) variation coefficient. The intra-group variation coefficient of the established real-time fluorescent quantitative PCR detection method is 0.27-1.50%, and the inter-group variation coefficient is 0.24-1.68%, which shows that the established real-time fluorescent quantitative PCR detection method has good repeatability.
6 clinical application
The real-time fluorescence quantitative PCR detection method of the established pigeon rotavirus A TaqMan probe is used for detecting 41 clinical detection pigeon source pathological materials, and the result shows that: 8 samples positive for fluorescence signal of FAM channel (Ct values of 21.01, 26.90, 21.80, 22.49, 19.76, 20.61, 22.57 and 24.45) show that the FAM channel is positive for pigeon rotavirus A, and the positive rate is 19.5 percent (8/41).
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Sequence listing
<110> animal husbandry and veterinary institute of agricultural academy of sciences of Fujian province
Primer, probe and kit for pigeon rotavirus A type fluorescent quantitative PCR detection
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ccgaactgct cctgtatgaa t 21
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cattgcccat tgctatccat tt 22
<210> 3
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
agtctgacat ccatttcatt gcacaacc 28
Claims (3)
1. A primer and a probe for pigeon rotavirus A type real-time fluorescent quantitative PCR detection are characterized in that: the primer sequences are as follows:
primer PiRVA-TF: 5'-CCGAACTGCTCCTGTATGAAT-3' the flow of the air in the air conditioner,
primer PiRVA-TR: 5'-CATTGCCCATTGCTATCCATTT-3', respectively;
the sequence of the probe is as follows:
probe PiRVA-probe: 5'-AGTCTGACATCCATTTCATTGCACAACC-3', and the 5 '-end is marked with a fluorescence reporter group FAM, and the middle and the 3' -end of the probe are respectively marked with a quenching group DBQ 1.
2. The use of the primers and probes as defined in claim 1 in the preparation of a kit for the detection of pigeon rotavirus type A specificity.
3. A pigeon rotavirus A type real-time fluorescent quantitative PCR detection kit is characterized in that: the kit comprises the primer and the probe of claim 1.
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| CN113046481A (en) * | 2021-03-30 | 2021-06-29 | 福建省农业科学院畜牧兽医研究所 | Primer, probe and kit for B-type fluorescence quantitative detection of pigeon adenovirus |
| CN113528706A (en) * | 2021-07-13 | 2021-10-22 | 苏州博腾生物制药有限公司 | Reagent for detecting replication-competent lentivirus by three-channel double-quenching probe and detection method thereof |
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| CN113046481A (en) * | 2021-03-30 | 2021-06-29 | 福建省农业科学院畜牧兽医研究所 | Primer, probe and kit for B-type fluorescence quantitative detection of pigeon adenovirus |
| CN113528706A (en) * | 2021-07-13 | 2021-10-22 | 苏州博腾生物制药有限公司 | Reagent for detecting replication-competent lentivirus by three-channel double-quenching probe and detection method thereof |
Non-Patent Citations (2)
| Title |
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| DENNIS RUBBENSTROTH等: "Identification of a novel clade of group A rotaviruses in fatally diseased domestic pigeons in Europe", 《TRANSBOUNDARY AND EMERGING DISEASES》, vol. 66, no. 1, pages 552 - 561 * |
| GENBANK: "Rotavirus A strain RVA/pigeon-wt/GER/GK-684/2017/G18P[17] isolate GK-684 glycoprotein (VP7) gene, complete cds", 《GENBANK》, pages 568764 * |
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