CN108315488B - Primer for identifying type of avian infectious bronchitis virus strain, RT-PCR detection kit, method and application - Google Patents
Primer for identifying type of avian infectious bronchitis virus strain, RT-PCR detection kit, method and application Download PDFInfo
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Abstract
The invention discloses a primer for identifying the type of an infectious bronchitis virus strain, an RT-PCR detection kit, a method and application, wherein the sequence of the primer is as follows: h120 upstream primer: 5'-ATTGCTTACGGTCCTCT-3', H120 downstream primer: 5'-CTGCTGGTTGACATCTT-3', respectively; QX upstream primer: 5'-GTACAGGGTCTTGTCCTA-3', QX downstream primer: 5'-GTGTTGCTTAATTCACCT-3', respectively; LDT3 upstream primer: 5'-CGCCACAGCAGGAAGAAT-3', LDT3 downstream primer: 5'-GTCCGTAGTTGGAATGAAGA-3', respectively; 4/91 upstream primer: 5'-CCAGATAGGCGGTGTTAG-3', 4/91 downstream primer: 5'-TCGGCAATAGGAAAGTGT-3' are provided. The invention has the beneficial effects that: the kit provided by the invention can be used for quickly identifying and diagnosing vaccine strains and epidemic wild strains of the infectious bronchitis only by amplifying four fragments with different sizes through one-time gradient RT-PCR, has the characteristics of high efficiency, low cost, strong specificity, high sensitivity and the like, and provides a convenient and quick detection method for identifying and diagnosing the infectious bronchitis viruses, analyzing molecular epidemiology and preventing and controlling the infectious bronchitis viruses.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a primer for identifying the type of an infectious bronchitis virus strain, an RT-PCR detection kit and application thereof, and is suitable for typing identification of common infectious bronchitis virus vaccine strains.
Background
Infectious bronchitis of chicken is an acute, highly contagious respiratory disease that occurs only in chickens, and is caused by infectious bronchitis virus, which is distributed worldwide. Every day of age is easy to feel, and the young chicken is characterized by breathing and breathing, and the egg yield and quality of the laying hens are reduced. The epidemic of infectious bronchitis causes huge economic loss to the poultry industry and seriously restricts the development of the poultry industry.
The avian infectious bronchitis virus belongs to the family of coronaviridae, the genus coronavirus, and the genome thereof has a high mismatching rate due to a unique transcription mechanism of a leader primer. Under the influence of immunization and the like, IBV is becoming variable at different rates and in different evolutionary directions, presenting complex genotypes and serotypes. Because the cross protection force between different serotype IBV strains is weak, the genetic variation is accelerated by the non-typing immunity, which brings great difficulty to the immune prevention of IB. Therefore, the type of the epidemic IBV strain needs to be accurately judged, and timely guidance is provided for the use of the vaccine.
The S protein of IBV is immunogenic and is hydrolyzed into two subunits, S1 and S2, where the S1 protein determines the serotype of IBV and affects the tissue phagocytosis of the virus. Serotyping of viruses requires a neutralization test, which is time consuming and laborious. Calvin L. Keeler (1998) et al performed comparative analyses of the S1 glycoprotein from 31 IBV strains from different regions to identify conserved and specific regions, and designed specific primers 6 for RT-PCR amplification consistent with the results of the serum neutralization experiments.
At present, H120 strain, LDT3-03 strain, 4/91 strain and popular QX strain are common vaccines on the market.
Disclosure of Invention
The invention aims to overcome the defects of conventional detection means, provides a primer for identifying the type of an infectious bronchitis virus strain, an RT-PCR detection kit, a method and application, and provides guidance for vaccine use.
In order to achieve the above object, the present invention provides a primer for identifying the type of an avian infectious bronchitis virus strain, which comprises a four-pair typing primer, wherein the sequence of the primer is as follows:
a first primer:
h120 upstream primer: 5'-ATTGCTTACGGTCCTCT-3' the flow of the air in the air conditioner,
h120 downstream primer: 5'-CTGCTGGTTGACATCTT-3', respectively;
and (2) primer II:
QX upstream primer: 5'-GTACAGGGTCTTGTCCTA-3' the flow of the air in the air conditioner,
QX downstream primer: 5'-GTGTTGCTTAATTCACCT-3', respectively;
primer three:
LDT3 upstream primer: 5'-CGCCACAGCAGGAAGAAT-3' the flow of the air in the air conditioner,
LDT3 downstream primer: 5'-GTCCGTAGTTGGAATGAAGA-3', respectively;
and (4) primer IV:
4/91 upstream primer: 5'-CCAGATAGGCGGTGTTAG-3' the flow of the air in the air conditioner,
4/91 downstream primer: 5'-TCGGCAATAGGAAAGTGT-3' are provided.
In order to better achieve the above object, the present invention provides an RT-PCR detection reagent for identifying the type of avian infectious bronchitis virus strain, which comprises the following components: RNA extraction reagent, reverse transcription reagent, PCR reaction reagent, four-pair typing primer and positive control sample;
the sequences of the primers are as follows:
a first primer:
h120 upstream primer: 5'-ATTGCTTACGGTCCTCT-3', and the adhesive tape is used for adhering the film to a substrate,
h120 downstream primer: 5'-CTGCTGGTTGACATCTT-3', respectively;
and (2) primer II:
QX upstream primer: 5'-GTACAGGGTCTTGTCCTA-3' the flow of the air in the air conditioner,
QX downstream primer: 5'-GTGTTGCTTAATTCACCT-3', respectively;
primer three:
LDT3 upstream primer: 5'-CGCCACAGCAGGAAGAAT-3', and the adhesive tape is used for adhering the film to a substrate,
LDT3 downstream primer: 5'-GTCCGTAGTTGGAATGAAGA-3', respectively;
and (4) primer IV:
4/91 upstream primer: 5'-CCAGATAGGCGGTGTTAG-3' the flow of the air in the air conditioner,
4/91 downstream primer: 5'-TCGGCAATAGGAAAGTGT-3' are provided.
Preferably, the concentration of the primer is 20. mu.M.
The RNA extraction reagent comprises: lysis solution, washing solution, magnetic beads, DEPC H 2 O；
The reverse transcription reagent comprises: buffer of 5 XM-MLV, dNTPs at 10mM, RRI at 40U/. mu.L, M-MLV at 200U/. mu.L, Random primer at 100. mu.M;
the PCR reaction reagent comprises: dd H 2 O, 2 XEx Taq PCR Mix (containing dNTPs);
the positive controls were: the reverse transcription products of RNA extracted from the allantoic fluid of H120 strain, LDT3-03 strain, 4/91 strain and epidemic QX virus inoculated chick embryo are used as positive control cDNA.
An RT-PCR detection kit for identifying the type of an infectious bronchitis virus strain is a kit containing the RT-PCR detection reagent.
The primer, the detection reagent or the kit can be applied to identification of infectious bronchitis virus live epidemics or preparation of detection products for identification of infectious bronchitis virus live epidemics.
In order to better achieve the above object, the present invention also provides an RT-PCR detection method for identifying the type of avian infectious bronchitis virus strain, the detection method comprising:
(1) extraction of sample RNA: taking 200. mu.L of sample, extracting RNA by using magnetic bead method reagent, and using 50. mu.L of DEPC H 2 Dissolving O to obtain total RNA;
(2) performing reverse transcription reaction by taking the total RNA obtained in the step (1) as a template to obtain a reverse transcription product sample cDNA;
(3) forming 8 reaction systems by using each pair of the four pairs of typing primers and respectively using the sample cDNA and the positive control sample cDNA obtained in the step (2) as templates to perform PCR reaction, and realizing gradient PCR amplification to obtain 8 PCR reaction solutions;
(4) and (4) carrying out agarose gel electrophoresis on the PCR reaction solution obtained in the step (3), and then judging the result according to the positive control of the band.
Wherein, the reverse transcription reaction system in the step (2) is as follows: taking 5.5 μ L of the total RNA obtained in the step (1), adding 8 μ L of 10mM dNTPs, 1.5 μ L of 100 μ M Random primer, 4 μ L of 5 XM-MLV Buffer, 0.5 μ L of 40U/. mu.L RRI, and 0.5 μ L of 200U/. mu.L M-MLV to 20 μ L, and mixing uniformly; the reverse transcription reaction conditions are as follows: 30 ℃ for 10min, 42 ℃ for 1h, and 70 ℃ for 5 min.
The PCR reaction systems in the step (3) are all as follows: mu.L of 2 XEx Taq PCR Mix (containing dNTPs), 1. mu.L of 20. mu.M upstream primer, 1. mu.L of 20. mu.M downstream primer, 2. mu.L of sample cDNA template or 2. mu.L of positive control cDNA template, plus 6. mu.L of dd H 2 O to 20 μ L; the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 35s, annealing at 55 ℃ or a gradient of 60 ℃ (where H120 and QX are 60 ℃ and LDT3 and 4/91 are 55 ℃) for 35s, extension at 72 ℃ for 45s, and total extension at 72 ℃ for 10min after a total of 32 cycles from the start of the denaturation.
The primer provided by the invention is designed by finding out a conserved region and a specific region through comparative analysis of genome sequences S1 of a plurality of strains of MASS, LDT3, 4/91, QX and other types of IBV published in GenBank.
In the invention, the size of a PCR product fragment obtained by amplifying the H120 primer is 447bp, the size of a PCR product fragment obtained by amplifying the QX primer is 829bp, the size of a PCR product fragment obtained by amplifying the LDT3 primer is 787bp, and the size of a PCR product fragment obtained by amplifying the 4/91 primer is 233 bp.
The beneficial effects of the invention are: IB serotypes are complex and diverse, the serological test identification is time-consuming and labor-consuming, and the invention can judge whether the vaccine contains H120, QX, LDT3, 4/91 and other strains or not only by one-time reverse transcription and one gradient PCR process and observing the size of a product fragment through electrophoresis; the kit provided by the invention can be used for quickly identifying and diagnosing vaccine strains and epidemic wild strains of the infectious bronchitis only by amplifying four fragments with different sizes through one-time gradient RT-PCR, has the characteristics of high efficiency, convenience, low cost, strong specificity, high sensitivity and the like, provides a convenient and quick detection method for identifying and diagnosing the infectious bronchitis viruses, analyzing molecular epidemiology and preventing and controlling the infectious bronchitis viruses, provides a basis for clinical vaccine screening, and has important significance for preventing and controlling IB.
Drawings
FIG. 1 is a diagram showing the result of PCR amplification of a pair of positive templates by primers in the kit; wherein, MK and 2000bp Marker are adopted; 1. H120, 2, QX, 3, LDT3, 4, 4/91.
FIG. 2 is a diagram showing the result of PCR amplification of two pairs of positive templates in the kit; wherein, MK and 2000bp Marker are adopted; 1. H120, 2, QX, 3, LDT3, 4, 4/91.
FIG. 3 is a diagram showing the result of PCR amplification of three pairs of positive templates with primers in the kit; wherein, MK and 2000bp Marker are adopted; 1. H120, 2, QX, 3, LDT3, 4, 4/91.
FIG. 4 is a diagram showing the result of PCR amplification of four pairs of positive templates with primers in the kit; wherein, MK and 2000bp Marker are adopted; 1. H120, 2, QX, 3, LDT3, 4, 4/91.
FIG. 5 is a diagram showing the results of electrophoresis of a bottle of commercial seedlings for detecting infectious bronchitis with the kit; wherein MK, 2000bp Marker, Y1 and sample H120 are amplified; p1, H120 positive control; n1, H120 negative control; y2, sample QX amplification; p2, QX positive control; n2, QX negative control; y3, sample LDT3 amplification; p3, LDT3 positive control; n3, LDT3 negative control; y4, sample 4/91 amplification; p4, 4/91 positive control; negative controls N4, 4/91.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1 primers
The embodiment of the invention provides a primer for identifying the type of an infectious bronchitis virus strain, which comprises a four-pair typing primer, wherein the sequence of the primer is as follows:
a first primer:
h120 upstream primer: 5'-ATTGCTTACGGTCCTCT-3' the flow of the air in the air conditioner,
h120 downstream primer: 5'-CTGCTGGTTGACATCTT-3';
and (2) primer II:
QX upstream primer: 5'-GTACAGGGTCTTGTCCTA-3' the flow of the air in the air conditioner,
QX downstream primer: 5'-GTGTTGCTTAATTCACCT-3', respectively;
primer three:
LDT3 upstream primer: 5'-CGCCACAGCAGGAAGAAT-3' the flow of the air in the air conditioner,
LDT3 downstream primer: 5'-GTCCGTAGTTGGAATGAAGA-3', respectively;
and (4) primer IV:
4/91 upstream primer: 5'-CCAGATAGGCGGTGTTAG-3' the flow of the air in the air conditioner,
4/91 downstream primer: 5'-TCGGCAATAGGAAAGTGT-3' are provided.
Example 2 detection reagent
The embodiment of the invention provides an RT-PCR detection reagent for identifying the type of an infectious bronchitis virus strain, which comprises the following components: RNA extraction reagent, reverse transcription reagent, PCR reaction reagent, four-pair typing primer and positive control sample;
wherein, the sequence of the primer is as follows:
a first primer:
h120 upstream primer: 5'-ATTGCTTACGGTCCTCT-3' the flow of the air in the air conditioner,
h120 downstream primer: 5'-CTGCTGGTTGACATCTT-3', respectively;
and (2) primer II:
QX upstream primer: 5'-GTACAGGGTCTTGTCCTA-3' the flow of the air in the air conditioner,
QX downstream primer: 5'-GTGTTGCTTAATTCACCT-3', respectively;
and (3) primer three:
LDT3 upstream primer: 5'-CGCCACAGCAGGAAGAAT-3', and the adhesive tape is used for adhering the film to a substrate,
LDT3 downstream primer: 5'-GTCCGTAGTTGGAATGAAGA-3', respectively;
and (4) primer IV:
4/91 upstream primer: 5'-CCAGATAGGCGGTGTTAG-3', and the adhesive tape is used for adhering the film to a substrate,
4/91 downstream primer: 5'-TCGGCAATAGGAAAGTGT-3' is added.
The concentration of the primers was 20. mu.M.
The RNA extraction reagent comprises: lysis solution, washing solution, magnetic beads, DEPC H 2 O；
The reagent for reverse transcription comprises: buffer of 5 XM-MLV, dNTPs at 10mM, RRI at 40U/. mu.L, M-MLV at 200U/. mu.L, Random primer at 100. mu.M;
the PCR reaction reagent comprises: dd H 2 O, 2 XEx Taq PCR Mix (containing dNTPs).
The positive controls were: the reverse transcription products of RNA extracted from allantoic fluid collected from H120 strain, LDT3-03 strain, 4/91 strain and popular QX virus inoculated chick embryo are used as positive control cDNA.
Example 3 detection kit
The embodiment of the invention provides an RT-PCR detection kit for identifying the type of an infectious bronchitis virus strain, and particularly provides a kit containing the RT-PCR detection reagent for identifying the type of the infectious bronchitis virus strain provided by the embodiment 2.
Example 4 detection method
The embodiment of the invention provides an RT-PCR detection method for identifying the type of an infectious bronchitis virus strain, which comprises the following steps:
(1) extraction of sample RNA: taking 200. mu.L of sample, extracting RNA by using magnetic bead method reagent, and using 50. mu.L of DEPC H 2 Dissolving O to obtain total RNA;
(2) performing reverse transcription reaction by taking the total RNA obtained in the step (1) as a template to obtain a reverse transcription product sample cDNA;
(3) forming 8 reaction systems by using each pair of the four pairs of typing primers and respectively using the sample cDNA and the positive control sample cDNA obtained in the step (2) as templates to perform PCR reaction, and realizing gradient PCR amplification to obtain 8 PCR reaction solutions;
(4) and (4) carrying out agarose gel electrophoresis on the PCR reaction solution obtained in the step (3), and then judging the result according to the positive control of the band.
Wherein, the reverse transcription reaction system in the step (2) is as follows: taking 5.5 μ L of the total RNA obtained in the step (1), adding 8 μ L of 10mM dNTPs, 1.5 μ L of 100 μ M Random primer, 4 μ L of 5 XM-MLV Buffer, 0.5 μ L of 40U/. mu.L RRI, and 0.5 μ L of 200U/. mu.L M-MLV to 20 μ L, and mixing uniformly; the reverse transcription reaction conditions are as follows: 30 ℃ for 10min, 42 ℃ for 1h, and 70 ℃ for 5 min.
The PCR reaction system of the step (3)All are as follows: mu.L of 2 XEx Taq PCR Mix (containing dNTPs), 1. mu.L of 20. mu.M upstream primer, 1. mu.L of 20. mu.M downstream primer, 2. mu.L of sample cDNA template or 2. mu.L of positive control cDNA template, plus 6. mu.L of dd H 2 O to 20 μ L; the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 35s, annealing at 55 ℃ or a gradient of 60 ℃ (where H120 and QX are 60 ℃ and LDT3 and 4/91 are 55 ℃) for 35s, extension at 72 ℃ for 45s, and extension at 72 ℃ for 10min after a total of 32 cycles from the start of denaturation.
Example 5 detection kit specificity verification
The embodiment of the invention carries out specificity verification on the kit provided by the embodiment 3, and the specific steps are as follows:
(1) extraction of positive control RNA: extracting four positive samples with magnetic bead method kit (200 μ L), and extracting with 50 μ L DEPC H 2 Dissolving O to obtain total RNA;
(2) performing reverse transcription reaction by taking the total RNA obtained in the step (1) as a template to obtain a reverse transcription product positive control sample cDNA;
the reaction system is as follows: taking 5.5 μ L of the total RNA obtained in the step (1), adding 8 μ L of 10mM dNTPs, 1.5 μ L of 100 μ M Random primer, 4 μ L of 5 XM-MLV Buffer, 0.5 μ L of 40U/. mu.L RRI, and 0.5 μ L of 200U/. mu.L M-MLV to 20 μ L, and mixing uniformly; the reverse transcription reaction conditions are as follows: 30 ℃ for 10min, 42 ℃ for 1h and 70 ℃ for 5 min.
(3) Taking the positive control sample cDNA obtained in the step (2) as a template, and respectively matching 4 individual systems with four pairs of typing primers to perform PCR reaction so as to realize gradient PCR amplification and obtain 4 PCR reaction solutions;
all 4 PCR reaction systems are: 10 μ L of 2 XEx Taq PCR Mix (containing dNTPs), 1 μ L of 20 μ M upstream primer, 1 μ L of 20 μ M downstream primer, 2 μ L of positive control cDNA template, plus 6 μ L of dd H 2 O to 20 μ L; the PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 35s, annealing at 55 ℃ or a gradient of 60 ℃ (where H120 and QX are 60 ℃ and LDT3 and 4/91 are 55 ℃) for 35s, extension at 72 ℃ for 45s, and total extension at 72 ℃ for 10min after a total of 32 cycles from the start of denaturation.
(4) And (3) carrying out agarose gel electrophoresis on the 4 PCR reaction solutions obtained in the step (3), as shown in figures 1-4, wherein the results show that the four pairs of primers have good specificity: the primer I only amplifies a band 447bp of the H120 strain, and other three strains are negative; the second primer only amplifies the QX strain to obtain a strip with a fragment size of 829bp, and other three strains are negative; the third primer is only used for amplifying LDT3 strain to obtain a band with the fragment size of 787bp, and other three strains are negative; 4/91 is only amplified by the fourth primer to obtain a band with the fragment size of 233bp, and the other three strains of viruses are negative.
Example 6 application
The embodiment of the invention provides a primer, a detection reagent or a detection kit, which is used for identifying the type of an infectious bronchitis virus strain, in particular to the identification of the strain of a clinical infectious bronchitis commercial vaccine. The method comprises the following specific steps:
(1) extraction of vaccine RNA
Freeze-dried seedlings were inoculated with 2 mL dd H 2 Diluting with O, mixing, extracting 200 μ L of the diluted solution, extracting RNA with Invitrogen RNA extraction kit, and extracting with 50 μ L of DEPC H 2 Dissolving O to obtain total RNA;
(2) reverse transcription reaction
Taking the total RNA obtained in the step (1) as a template to perform reverse transcription reaction, wherein the reaction system is as follows: taking 5.5 μ L of the total RNA obtained in the step (1), adding 8 μ L of 10mM dNTPs, 1.5 μ L of 100 μ M Random primer, 4 μ L of 5 XM-MLV Buffer, 0.5 μ L of 40U/. mu.L RRI, and 0.5 μ L of 200U/. mu.L M-MLV to 20 μ L, and mixing uniformly; the reverse transcription reaction conditions are as follows: obtaining a reverse transcription product sample cDNA by multiplying by 10min at 30 ℃, multiplying by 1h at 42 ℃ and multiplying by 5min at 70 ℃;
(3) PCR reaction
Forming 8 reaction systems by using each pair of the four pairs of typing primers and taking the sample cDNA obtained in the step (2) and the positive control sample cDNA as templates respectively to perform PCR reaction, and realizing gradient PCR amplification to obtain 8 PCR reaction solutions;
the PCR reaction systems of the step (3) are all as follows: 10 μ L of 2 XEx Taq PCR Mix (containing dNTPs), 1 μ L of 20 μ M upstream primer, 1 μ L of 20 μ M downstreamPrimer, 2. mu.L sample cDNA template or 2. mu.L positive control cDNA template, plus 6. mu.L dd H 2 O to 20 μ L; the PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 35s, annealing at 55 ℃ or a gradient of 60 ℃ (where H120 and QX are 60 ℃ and LDT3 and 4/91 are 55 ℃) for 35s, extension at 72 ℃ for 45s, and total extension at 72 ℃ for 10min after a total of 32 cycles from the beginning of denaturation;
(4) carrying out agarose gel electrophoresis on the PCR reaction solution obtained in the step (3), and then judging the result according to the positive control of the band; as shown in FIG. 5, the PCR products obtained by the four primer pairs have two bands at the primers H120 and LDT3, and the sizes of the two bands are consistent with the expected sizes of the primers H120 and LDT3, respectively, and the results are consistent with the labeling of vaccine manufacturers.
The reagents, materials and the like used in the above examples are all available from Biochemical Co., Ltd unless otherwise specified
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent replacements, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (1)
1. An RT-PCR detection kit for identifying the type of an infectious bronchitis virus strain, which is characterized by comprising: RNA extraction reagent, reverse transcription reagent, PCR reaction reagent, four-pair typing primer and positive control sample;
the four pairs of typing primers have the following sequences:
the upstream primer of the H120 is 5'-ATTGCTTACGGTCCTCT-3',
5'-CTGCTGGTTGACATCTT-3' as downstream primer of H120;
the upstream primer of QX is 5'-GTACAGGGTCTTGTCCTA-3',
5'-GTGTTGCTTAATTCACCT-3' as QX downstream primer;
an upstream primer of LDT3, 5'-CGCCACAGCAGGAAGAAT-3',
5'-GTCCGTAGTTGGAATGAAGA-3' as the downstream primer of LDT3;
4/91, an upstream primer of 5'-CCAGATAGGCGGTGTTAG-3',
4/91 downstream primer 5'-TCGGCAATAGGAAAGTGT-3';
the concentration of the four pairs of typing primers is 20 mu M;
the RNA extraction reagent comprises: lysis solution, washing solution, magnetic beads and DEPC treatment water;
the reverse transcription reagent comprises: 5 XM-MLV buffer, 10mM dNTPs, 40U/. mu.L RNase inhibitor, 200U/. mu.L M-MLV reverse transcriptase and 100. mu.M random primer;
the PCR reaction reagent comprises ddH 2 O , 2×Ex Taq PCR Mix；
The positive controls were: respectively inoculating H120 strain, LDT3-03 strain, 4/91 strain and popular QX seed virus to chick embryos, collecting allantoic fluid and extracting reverse transcription products of RNA;
the using method of the kit comprises the following steps:
(1) extraction of sample RNA: taking 200 mu L of sample, extracting RNA by using a magnetic bead method reagent, and dissolving with 50 mu L of DEPC (diethyl phthalate) treated water to obtain total RNA;
(2) performing reverse transcription reaction by taking the total RNA obtained in the step (1) as a template to obtain a reverse transcription product sample cDNA;
(3) forming 8 reaction systems by using each pair of the four pairs of typing primers and respectively using the sample cDNA and the positive control sample cDNA obtained in the step (2) as templates to perform PCR reaction, and realizing PCR amplification to obtain 8 PCR reaction solutions;
(4) carrying out agarose gel electrophoresis on the PCR reaction solution obtained in the step (3), and then judging the result according to the positive control of the strip;
the reverse transcription reaction system in the step (2) is as follows: taking 5.5 mu L of the total RNA obtained in the step (1), adding 8 mu L of 10mM dNTPs, 1.5 mu L of 100 mu M random primer, 4 mu L of 5 xM-MLV buffer solution, 0.5 mu L of 40U/mu L RNase inhibitor and 0.5 mu L of 200U/mu L M-MLV to 20 mu L, and uniformly mixing; the reverse transcription reaction conditions are as follows: 10min at 30 ℃, 1h at 42 ℃ and 5min at 70 ℃;
the PCR reaction system in the step (3) is: 10 μ L of 2 XEx Taq PCR Mix, 1 μ L of 20 μ M upstream primer, 1 μ L of 20 μ M downstream primer, 2 μ L of sample cDNA template or 2 μ L of positive control cDNA template, plus 6 μ L of dd H 2 O to 20. mu.L(ii) a The conditions of the PCR reaction are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 35 s; annealing at 55 ℃ or 60 ℃ for 35s, extension at 72 ℃ for 45s, and performing 32 cycles, wherein the temperature of H120 and QX is 60 ℃, the temperature of LDT3 and 4/91 is 55 ℃, and the extension at 72 ℃ is 10 min.
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| CN108866242A (en) * | 2018-08-24 | 2018-11-23 | 扬州大学 | For identifying the half Nest RT-PCR serotype specific primer and kit of different serotypes infectious bronchitis virus live vaccine |
| CN110747292A (en) * | 2019-11-28 | 2020-02-04 | 青岛易邦生物工程有限公司 | Method for identifying avian infectious bronchitis QX type virus wild virus and vaccine virus |
| CN112553376B (en) * | 2020-12-23 | 2023-06-02 | 四川大学 | Primer group, kit, multiple PCR detection method for distinguishing different IBV vaccine strains and application of multiple PCR detection method |
| CN116024388A (en) * | 2023-01-05 | 2023-04-28 | 福建圣泽生物科技发展有限公司 | RT-PCR detection kit for infectious bronchitis virus GVI-1 genotype and application thereof |
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