CN108950068B - Kit for identifying and detecting QX-type strains of chicken infectious bronchitis viruses - Google Patents

Abstract

The invention relates to the technical field of biological detection, and aims to provide a kit for identifying and detecting chicken infectious bronchitis virus QX type strains. The kit comprises two pairs of primers, namely a universal primer for amplifying the infectious bronchitis virus and a primer for specifically amplifying the QX genotype strain; wherein, the sequence of the primer for specifically amplifying the QX genotype strain is shown as SEQ ID NO: 5 and SEQ ID NO: and 6. The invention establishes an effective RT-PCR detection method by designing the specific primer of the QX genotype, and can achieve the aim of carrying out typing detection on the IBV of the QX genotype only by one RT-PCR reaction. The kit adds 2 pairs of primers into the same PCR system under the same PCR reaction condition, not only can effectively detect IBV in a clinical sample or chick embryo allantoic fluid, but also can specifically distinguish the IBV of the QX genotype. The detection method is rapid, efficient and accurate, and has great significance for guiding the selection of vaccines and the rapid prevention and control of IB.

Description

Kit for identifying and detecting QX-type strains of chicken infectious bronchitis viruses

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a kit for identifying and detecting a QX strain of avian infectious bronchitis virus.

Background

Infectious Bronchitis (IB) is an acute, highly contagious disease caused by Infectious Bronchitis Virus (IBV). The disease can affect respiratory system, urogenital system and digestive system of chicken. Is one of the important infectious diseases seriously harming the poultry industry in the world.

The avian infectious bronchitis virus belongs to the genus coronavirus of the family Coronaviridae of the order Nidovirales. Its genome is an unfragmented positive-strand RNA virus, approximately 27.6kb in length. The viral genome is 5 '-1 a-1b-S1-S2-3a-3b-3c-M-5a-5b-N-poly (A) -3' from 5 'end to 3' end in sequence. IBV contains 4 major structural proteins: spike protein (S), membrane protein (M), nucleocapsid protein (N), and vesicle membrane protein (E). Most of the antigenic determinants of IBV are located in the S protein, which not only mediates fusion of the virus with susceptible cells, determines the virulence of the virus, but also determines the antigenicity of the virus. In the transcription process of the IBV, the unique transcription mechanism of the same leader sequence to transcribe different segments is used, and the RNA enzyme deficiency proofreading mechanism is added, so that the genome of the IBV is easy to have point mutation, deletion, insertion and recombination, finally, the variation of an IBV strain is caused, and new serotypes and genotypes are continuously generated. A great deal of research shows that the S1 gene of IBV is the gene with the highest mutation frequency and mutation degree, and IBV typing and genetic evolution analysis based on the S1 gene become a great research hotspot in recent years.

The various IBVs can be classified into strains of different genotypes by phylogenetic analysis of the S1 gene of IBV. Currently, IBV strains of the QX genotype are prevalent in China. IBV QX strains were previously circulating in 1990s only in china, and have been widely circulating after 2000s around the world including europe and russia. Conventional vaccines do not provide protection against infection by QX strains. The prevalence of QX strains therefore represents a significant economic loss to the chicken industry. Clinically, the specificity and rapid differential diagnosis of the IBV strain of the QX genotype are urgently needed so as to guide the selection of the vaccine in time and reduce the loss caused by the IBV infection.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects in the prior art and provides a kit for identifying and detecting the QX type strains of the avian infectious bronchitis viruses.

In order to solve the technical problem, the solution of the invention is as follows:

provides a kit for identifying and detecting chicken infectious bronchitis virus QX type strains, which comprises the following two pairs of primers: universal primers for amplifying infectious bronchitis viruses and primers for specifically amplifying QX genotype strains; the sequence is as follows:

general purpose-F: 5'-CCAAAGTGCCTTCAGACC-3' (shown as SEQ ID NO: 3)

General formula-R: 5'-GCTAGACCAAGCCATACC-3' (shown as SEQ ID NO: 4)

QX-F: 5'-GTCATTTCTGAGTCAGTTTGTG-3' (shown as SEQ ID NO: 5)

QX-R: 5'-TGCGTTAATACTAAGGGCTC-3' (shown in SEQ ID NO: 6).

In the invention, the kit comprises the following components:

(1) RNA extraction reagent: trizol reagent;

(2) reverse transcription reagent: 5 times reaction buffer solution, 10mM dNTP Mix, 0.2. mu.g/. mu.l random primer, 20U/. mu.l RNase inhibitor, 200U/. mu.l reverse transcriptase and clean-free water;

(3) PCR reaction reagent: 2 XTaq Master Mix, sterilized double distilled water;

(4) typing primers: the concentrations of the universal primer and the QX genotype strain specific primer are both 10 mu M;

(5) positive control sample: cDNA of reverse transcription product of nucleic acid extract of QX genotype strain;

(6) negative control samples: extracting reverse transcription product cDNA of RNA from allantoic fluid of normal chick embryo.

In the invention, the positive control sample is a reverse transcription product cDNA of allantoic fluid extracted RNA of a QX genotype strain IBV-HF infected chick embryo.

The kit provided by the invention comprises the following steps:

(1) extracting total RNA from a swab to be detected, a tissue or an allantoic fluid sample infected with chick embryos:

adding 500 μ l of the treated swab, tissue or infected chick embryo allantoic fluid sample (less than 500 μ l can be made up to 500 μ l with sterile PBS), adding 500 μ l of Trizol reagent, mixing, and standing at room temperature for 5 min; adding 200 μ l chloroform, mixing for 15s, standing at room temperature for 5 min; centrifuging at 12000rpm for 10min at 4 deg.C; taking supernatant (400-; centrifuging at 12000rpm for 10min at 4 deg.C; discarding the supernatant, adding 1ml of 75% ethanol, centrifuging at 4 ℃ and 12000rpm for 7 min; the supernatant was discarded, the precipitate was dried for 10-15min, and 10. mu.l of nucleic-free water was added to dissolve it, to obtain total RNA. (Note: the tip and EP tube used for RNA extraction should not contain RNase)

(2) Taking the total RNA extracted in the previous step as a template to perform reverse transcription reaction. The reaction system is as follows: mu.l total RNA template, 4. mu.l 5 × reaction buffer, 2. mu.l 10mM dNTP Mix, 1. mu.l 0.2. mu.g/. mu.l random primer, 1. mu.l 20U/. mu.l RNase inhibitor, 1. mu.l 200U/. mu.l reverse transcriptase, in total 20. mu.l. After mixing evenly, the mixture reacts for 1h at the constant temperature of 42 ℃, and then the reaction is stopped for 10min at the constant temperature of 70 ℃ to obtain cDNA.

(3) And (2) performing PCR amplification by using the reverse transcription product cDNA as a template and two pairs of typing primers, wherein the PCR system comprises: 2 XTaq Master Mix 12.5. mu.l, 10. mu.M universal upstream and downstream primers (Universal-F, Universal-R) each 0.8. mu.l, 10. mu.M QX genotype primers (QX-F, QX-R) each 0.4. mu.l, cDNA template 1. mu.l, sterile double distilled water (ddH)₂O) to 25. mu.l.

The reaction conditions of the PCR reaction system are as follows: pre-denaturation at 95 ℃ for 5min, then performing cycles of denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 30s, wherein the cycles are 35, extension at 72 ℃ for 10min and extension at 16 ℃ for 5 min;

(4) and (3) carrying out 1% agarose gel electrophoresis on the PCR reaction solution, and judging the result after EB (Epstein-Barr) dyeing.

In the invention, two bands of about 200bp and 340bp are obtained by the RT-PCR amplification of the QX genotype IBV strain, and only one band of about 200bp is obtained by the amplification of the non-QX strain. PCR products obtained by amplification of the QX strain IBV-HF are two nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention establishes an effective RT-PCR detection method by designing the specific primer of the QX genotype. The purpose of carrying out typing detection on the IBV of the QX genotype can be achieved only by one RT-PCR reaction.

2. The kit adds 2 pairs of primers into the same PCR system under the same PCR reaction condition, not only can effectively detect IBV in a clinical sample or chick embryo allantoic fluid, but also can specifically distinguish the IBV of the QX genotype.

3. The detection method is rapid, efficient and accurate, and has great significance for guiding the selection of vaccines and the rapid prevention and control of IB.

Drawings

FIG. 1 shows the results of RT-PCR amplification of IBV-HF strains (QX type) and M41 (non-QX type) by the kit.

In the figure, M.100bp marker; 1. negative control; m41; IBV-HF

FIG. 2 shows the result of specific amplification of IBV by RT-PCR in the kit.

In the figure, M.100bp marker; 1. negative control; NDV, AIV H5 subtype, AIV H7 subtype, AIV H9 subtype, IBDV; m41; IBV-HF.

FIG. 3 is the result of the test of the selected strain by the kit.

In the figure, M.100bp marker; 1. negative control; NDV, AIV H5 subtype, AIV H7 subtype, AIV H9 subtype, IBDV; IBV-GX-1023(LDT-3 type); 8.4/91; 9-14.M41, H120, H52, ZJ971, JH06011, JH06111(Mass type); IBV-SD, IBV-QX-H120, IBV-GX-0808, IBV-HF (QX type).

Detailed Description

The kit used in the invention comprises the following components:

(1) RNA extraction reagent: trizol reagent;

(2) reverse transcription reagent: 5 × reaction buffer, 10mM dNTP Mix, 0.2 μ g/μ l random primer, 20U/μ l RNase inhibitor, 200U/μ l reverse transcriptase, and clean-free water.

(3) PCR reaction reagent: 2 × Taq Master Mix, sterilized double distilled water.

(4) Typing primers: the concentrations of the universal primer and the QX genotype strain specific primer (shown as SEQ ID NO: 3-6) are both 10 mu M;

(5) positive control sample: cDNA of reverse transcription product of nucleic acid extract of QX genotype strain;

(6) negative control samples: extracting reverse transcription product cDNA of RNA from allantoic fluid of normal chick embryo.

The using method of the kit sequentially comprises the following steps:

(1) extracting total RNA from a swab to be detected, a tissue or an allantoic fluid sample infected with chick embryos:

adding 500 μ l of treated swab, tissue or infected chick embryo allantoic fluid sample (less than 500 μ l can be diluted with sterile PBS) into 1.5ml EP tube, adding 500 μ l Trizol reagent, mixing, and standing at room temperature for 5 min; adding 200 μ l chloroform, mixing for 15s, standing at room temperature for 5 min; centrifuging at 12000rpm for 10min at 4 deg.C; taking supernatant (400-; centrifuging at 12000rpm for 10min at 4 deg.C; discarding the supernatant, adding 1ml of 75% ethanol, centrifuging at 4 ℃ and 12000rpm for 7 min; the supernatant was discarded, the precipitate was dried for 10-15min, and 10. mu.l of nucleic-free Water was added to dissolve it to obtain total RNA. (Note: the tip used for RNA extraction should not contain RNase in the EP tube)

(2) Taking the total RNA extracted in the previous step as a template to perform reverse transcription reaction, wherein the system is as follows: mu.l total RNA template, 4. mu.l 5 × reaction buffer, 2. mu.l 10mM dNTP Mix, 1. mu.l 0.2. mu.g/. mu.l random primer, 1. mu.l 20U/. mu.l RNase inhibitor, 1. mu.l 200U/. mu.l reverse transcriptase, in total 20. mu.l. After mixing evenly, the mixture reacts for 1h at the constant temperature of 42 ℃, and then the reaction is stopped for 10min at the constant temperature of 70 ℃ to obtain cDNA.

(3) And (2) performing PCR amplification by using the reverse transcription product cDNA as a template and two pairs of typing primers, wherein the PCR system comprises: 2 XTaq Master Mix 12.5. mu.l, 10. mu.M universal upstream and downstream primers (Universal-F, Universal-R) each 0.8. mu.l, 10. mu.M QX genotype primers (QX-F, QX-R) each 0.4. mu.l, cDNA template 1. mu.l, sterile double distilled water (ddH)₂O) to 25. mu.l.

The reaction conditions of the PCR reaction system are as follows: pre-denaturation at 95 ℃ for 5min, then entering the cycle of denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 30s, and carrying out 35 cycles in total, extension at 72 ℃ for 10min and extension at 16 ℃ for 5 min;

(4) and (3) electrophoretic detection of an amplification product: and (3) carrying out agarose gel electrophoresis on the reaction product, spotting 5 microliter of the amplification product into 1% agarose gel, carrying out 250V electrophoresis for 25min, and observing by a gel imaging system. Two bands of about 200bp and 340bp are obtained by the RT-PCR amplification of the QX genotype strains, and only one band of about 200bp is obtained for the non-QX genotype strains.

(5) Sequencing of amplification products: and (3) taking the specific band obtained by amplification of the positive control strain IBV-HF to send to a sequencing company for sequencing determination. The PCR product obtained by amplification is two nucleotide sequences shown in SEQ ID NO1-2, and the sizes are 193bp and 339bp respectively;

(6) the designed method can be used for amplifying other QX genotype or non-QX genotype strains to obtain similar results: about 200bp and about 340bp bands are obtained by amplifying the QX genotype strains, and only about 200bp band is obtained when the non-QX genotype strains are amplified.

The following is a further description with reference to the examples and the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

EXAMPLE 1 RNA extraction

1 test sample treatment

Treatment of tissue samples: tissues such as trachea, kidney and the like of dead chickens, which are easy to separate IBV, are cut into pieces by scissors and then are fully ground by adding sterilized PBS according to the volume ratio of 1: 3. The homogenized tissue suspension was repeatedly frozen and thawed at-20 deg.C (or lower) to room temperature for 3 times, centrifuged at 12,000rpm for 10min, and 200. mu.l of the supernatant was taken in a fresh sterilized centrifuge tube.

Processing of swab samples: fresh throat or cloaca swabs were added to 1ml of sterile PBS and after shaking to homogenize, the swabs were discarded (the liquid in the swabs was collected as completely as possible). The leachate was centrifuged at 12,000rpm for 10min and 200. mu.l of the supernatant was taken in a fresh sterilized centrifuge tube.

Allantoic fluid treatment: virus-infected chick embryo allantoic fluid was collected and centrifuged at 12,000rpm for 10min, and 200. mu.l of the supernatant was taken in a fresh sterilized centrifuge tube.

2 viral RNA extraction

Adding 500 μ l of treated swab, tissue or infected chick embryo allantoic fluid sample into 1.5ml EP tube, adding 500 μ l of Trizol reagent, mixing, and standing at room temperature for 5 min; adding 200 μ l chloroform, mixing for 15s, standing at room temperature for 5 min; centrifuging at 12000rpm for 10min at 4 deg.C; adding equal volume of isopropanol into 500 μ l of the supernatant, slightly mixing, and standing at room temperature for 5 min; centrifuging at 12000rpm for 10min at 4 deg.C; discarding the supernatant, adding 1ml of 75% ethanol, centrifuging at 4 ℃ and 12000rpm for 7 min; the supernatant was discarded, the remaining liquid in the tube was removed immediately and carefully blotted out (taking care that the tip did not touch the pellet), the EP tube was air dried on clean paper for 15min with an opening, and 10. mu.l of nucleic-free water was added to dissolve it to obtain total RNA. (Note: the tip and EP tube used for RNA extraction should not contain RNase)

EXAMPLE 2 reverse transcription reaction (RT reaction)

(4) Taking the total RNA extracted in the previous step as a template to perform reverse transcription reaction, wherein the reverse transcription reaction system comprises:

mu.l total RNA template, 4. mu.l 5 × reaction buffer, 2. mu.l 10mM dNTP Mix, 1. mu.l 0.2. mu.g/. mu.l random primer, 1. mu.l 20U/. mu.l RNase inhibitor, 1. mu.l 200U/. mu.l reverse transcriptase, in total 20. mu.l. After mixing evenly, the mixture reacts for 1h at the constant temperature of 42 ℃, and then the reaction is stopped for 10min at the constant temperature of 70 ℃ to obtain cDNA. Stored at-20 deg.C

EXAMPLE 3 PCR reaction

1. Primer design

The S1 gene sequences of IBV QX strains and some classical QX strains which are popular in China in recent years in GeneBank are collected, and the total number is 212. Meanwhile, 49 non-QX strains with different genotypes are selected from GeneBank, and the number of S1 gene sequences is 49. The 261S 1 genes were subjected to sequence alignment using DNAMAN software, and 1 pair of primers (QX-F, QX-R) were designed from a conserved region of the S1 gene of the QX-type strain using Primer design software Premier Primer 5, and were not conserved on strains other than the QX-type strain. General primers (general-F, general-R, Table 1) were referred to the Master thesis in Lingyi. The primers are synthesized by Hangzhou Optingke biotechnology Limited and purified by PAGE. The primer information is shown in Table 1.

TABLE 1 kit typing primer information

2. Positive standard preparation

IBV-HF (QX type) strain is inoculated to SPF chick embryos of 9-11 days old, and allantoic fluid of infected chick embryos is harvested 36-72h after the chick embryos are infected. After the chick embryo allantoic fluid is processed by the method, total RNA is extracted from the allantoic fluid and is reversely transcribed into cDNA.

The cDNA concentration was then determined and stored at-20 ℃ until use.

Optimization of PCR reaction System

The cDNA prepared as described above was used as a template to optimize conditions such as primer concentration, annealing temperature, extension time, PCR cycle number, etc. in the PCR program.

The results of the optimization of the reaction system and the amplification procedure were as follows:

in a 25 μ LPCR reaction system2 XTaq Master Mix 12.5. mu.l, Universal upstream and downstream primers (Universal-F, Universal-R) each 0.8. mu.l, QX genotype primers (QX-F, QX-R) each 0.4. mu.l, cDNA template 1. mu.l, ddH₂O 9.1μl。

The reaction conditions of the PCR reaction system are as follows: pre-denaturation at 95 ℃ for 5min, then entering the cycle of denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 30s, wherein the cycle is 35 cycles, extension at 72 ℃ for 10min and extension at 16 ℃ for 5 min;

the PCR amplification result of the positive standard by using the optimized system is shown in figure 1, two bands of about 200bp and 340bp are amplified by a QX type IBV strain (IBV-HF), and only one band of about 200bp is amplified by a non-QX type IBV strain (M41) (figure 1). The amplification results were consistent with the expected design.

PCR product sequencing validation

And (3) recovering a PCR product obtained by amplifying the positive standard substance, sending the PCR product to a sequencing company for sequencing analysis, verifying that the amplified sequence is completely consistent with an expected theoretical sequence, and displaying that the amplified sequence is a corresponding fragment through BLAST analysis. The sequence of the specific strain amplification fragment is shown as SEQ ID NO 1-2. Sequencing verification is carried out on the bands of about 340bp of other 4 QX genotype strains, and Blast analysis shows that the sequences of the strains have extremely high similarity with the QX genotype strains.

EXAMPLE 4 specificity verification of kit

Selecting common avian viruses including Avian Influenza Virus (AIV) H5, H7 and H9 subtype viruses, chicken Newcastle Disease Virus (NDV) and chicken embryo allantoic fluid or allantoic membrane infected by Infectious Bursal Disease Virus (IBDV), extracting RNA, performing reverse transcription to obtain cDNA, and performing PCR detection by using a system optimized by an IBV typing detection kit. As shown in FIG. 2, the cDNA of the positive control IBV-HF strain amplified a specific target band, and the non-QX strain also amplified a target band, whereas no band could be amplified for the common avian viruses AIV (subtypes H5, H7 and H9), NDV and IBDV (FIG. 2).

Example 5 detection and verification of samples of different genotype strains

Partial strains stored in animal virology key laboratories of the department of agriculture of Zhejiang university and partial strain cDNA awarded by the poultry disease research laboratory of the animal science and technology college of Guangxi university are taken as verification objects. The strains verified include 4/91 type strains, LDT-3 type strains (IBV-GX-1023), Mass type strains (M41, H120, H52, ZJ971, JH06011, JH06111), QX type strains (IBV-SD, IBV-QX-H120, IBV-GX-0808, IBV-HF) and common avian viruses AIV (subtype H5, H7, H9), NDV, IBDV. Nucleic acid extracts of allantoic fluid of chicken embryos infected by the strains are respectively detected by the kit, and the results show that DNA bands of about 200bp and 340bp are amplified from QX genotype IBV strains, a band of about 200bp is amplified from non-QX IBV strains, and a band is not amplified from non-IBV strains (figure 3). The specificity and the effectiveness of the IBV RT-PCR typing detection kit are verified.

Sequence listing

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taaggacgtc tataatcaaa gtgcggcttc catagctatg acagcacctc ctcagggtat 180

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Claims (2)

1. A kit for identifying and detecting chicken infectious bronchitis virus QX type strains is characterized by comprising two pairs of primers, namely a universal primer for amplifying the infectious bronchitis virus and a primer for specifically amplifying the QX genotype strains; wherein, the sequence of the primer for specifically amplifying the QX genotype strain is as follows:

QX-F：5'-GTCATTTCTGAGTCAGTTTGTG-3'

QX-R：5'-TGCGTTAATACTAAGGGCTC-3'

the sequence of the universal primer for amplifying the infectious bronchitis virus is as follows:

general purpose-F: 5'-CCAAAGTGCCTTCAGACC-3'

General formula-R: 5'-GCTAGACCAAGCCATACC-3' are provided.

2. The kit according to claim 1, characterized in that it comprises the following components:

(1) RNA extraction reagent: trizol reagent;

(2) reverse transcription reagent: 5 multiplied by reaction buffer solution, 10mM dNTP Mix, 0.2 mug/mug random primer, 20U/mug RNase inhibitor, 200U/mug reverse transcriptase and clean-free water;

(3) PCR reaction reagent: 2 XTaq Master Mix, sterilized double distilled water;

(4) typing primers: the concentrations of the universal primer and the QX genotype strain specific primer are both 10 mu M;

(5) positive control sample: cDNA of reverse transcription product of nucleic acid extract of QX genotype strain;

(6) negative control samples: extracting reverse transcription product cDNA of RNA from allantoic fluid of normal chick embryo;

the positive control sample is cDNA of a reverse transcription product of allantoic fluid extracted RNA of a chicken embryo infected by a QX genotype strain IBV-HF.

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