CN106636457A - Kit for detecting 4/91-type infectious bronchitis viruses - Google Patents

Abstract

The invention provides a kit for detecting 4/91 type infectious bronchitis virus, belonging to the technical field of RT-PCR detection. The kit of the present invention contains a pair of specific primers, the nucleotide sequences of which are respectively shown in SEQ ID NO.1‑2. The invention also provides a method for detecting 4/91 infectious bronchitis virus. The kit and the detection method of the invention have the advantages of accurate detection, high sensitivity, strong specificity, simplicity and speed, and good clinical sample detection ability.

Description

A kit for detecting 4/91 type infectious bronchitis virus

technical field

The invention relates to the field of molecular biology, in particular to a kit for detecting 4/91 type infectious bronchitis virus and its application.

Background technique

Infectious bronchitis virus (IBV) belongs to the Coronaviridae family and belongs to the genus Coronavirus. It is an enveloped single-stranded positive-sense RNA virus with a genome length of about 27.6 kb. The IBV genome encodes four essential structural proteins: spike protein (S), membrane protein (M), nucleocapsid protein (N) and small membrane protein (E). Spike protein and membrane protein are the main surface proteins, which are located on the outermost envelope of the virion and are the main components of the coronavirus fibrils. The nucleotide sequence variation of S gene among different strains of IBV is relatively large, some can reach more than 50%, and the amino acid variation of S protein of different IBV strains mostly occurs on S1. S1 protein is also the main protein of IBV serotype-specific epitope, which has a certain influence on the tissue tropism and virulence of IBV, and can induce the body to produce specific antibodies.

Due to the discontinuity of IBV genome RNA replication and the incomplete proofreading mechanism of RNA polymerase, the IBV genome is very prone to mutations, deletions, insertions, and homologous recombination between genomes of different strains, resulting in many IBV serotypes and new IBV strains. Serotypes are also emerging, but QX-like type IBV is the main epidemic strain in the world, and the 4/91 type IBV vaccine has a good cross-protection effect on this epidemic strain, so it is often necessary to control the 4/91 type IBV in the vaccine during production. 91 IBV strains are identified to determine whether the vaccine contains 4/91 IBV strains to ensure the immune effect of the vaccine. Therefore, the specific identification of 4/91 type IBV is a key link in the prevention and control of this disease. A variety of IBV diagnostic methods have been established, such as neutralization test, ELISA, hemagglutination and hemagglutination inhibition test, etc. Although these methods are conventional techniques, they are specifically applied to IBV detection, or due to the large differences in antigens between IBV serotypes The sensitivity is reduced, resulting in missed detection; or false positives due to the defects of conventional serological methods themselves, resulting in false detection. The traditional virus isolation method has high requirements on the timing of collecting disease materials and the freshness of disease materials. It is necessary to inoculate 9-11-day-old chicken embryos for subculture to observe embryonic changes, and to conduct chicken embryo neutralization tests with various standard positive sera , and the detection cycle often takes more than a few weeks, which has obvious limitations.

RT-PCR technology is a rapid, sensitive and specific molecular biological detection method. The emergence of this technology in the 1980s provided a new option for virus detection, and it has been widely used in the detection of various viruses. The sequence homology among many serotypes of IBV is high, and new mutant strains are constantly produced, which brings troubles to the rapid diagnosis of IBV serotypes.

Contents of the invention

The first object of the present invention is to provide a pair of specific primers for detecting IBV 4/91.

The second object of the present invention is to provide a kit for detecting 4/91 type infectious bronchitis virus.

Considering the high sequence homology among many serotypes of IBV, it is a technical difficulty in this field to find the differences among the serotypes, determine the conserved sequences of each difference, and select the target region from the hypervariable region of the S1 gene. The present invention refers to GenBank Specific primers were designed and screened for the hypervariable region of the registered 4/91 type IBV-S1 gene sequence (GenBank accession number KF377577). IBV-S1 protein is the most variable structural protein of IBV. Among the many alternative primers, after multiple screenings and comparison tests to exclude possible non-specific matches between the primers and the sequences of other species, an optimized primer pair was finally obtained.

The preferred specific primer pair provided by the present invention has a sequence of:

Upstream primer 5'-TCTGATTGCACTGCTGGTACT-3' (SEQ ID NO.1); and downstream primer 5'-GCACCCGTAACGTGAGTTG-3' (SEQ ID NO.2).

The upstream primer is between 20500nt-20520nt of GenBank accession number KF377577, and the downstream primer is between 20858nt-20876nt. After extracting total RNA from the sample and performing reverse transcription (RT) to synthesize cDNA, the above primers were used to perform polymerase chain reaction (PCR). ℃ 45s, 72℃ 25s), and finally extended at 72℃ for 10min, the PCR product was analyzed by agarose gel electrophoresis, and the target band was detected by a UV gel imager. If the target band was amplified, it was proved to be 4/91 Type IBV test positive, otherwise negative.

The invention provides the application of the above-mentioned pair of specific primers in preparing a kit for identifying infectious bronchitis virus serotypes.

The invention provides the application of the above-mentioned pair of specific primers in the preparation and detection of 4/91 type infectious bronchitis virus kit.

The detection reagent containing the specific primer pair shown in SEQ ID NO.1-2 belongs to the protection scope of the present invention.

The kit containing the specific primer pair shown in SEQ ID NO.1-2 belongs to the protection scope of the present invention.

Further, the working program of the PCR stage of the kit of the present invention is: 94°C for 5 minutes; 94°C for 45s, 58°C for 45s, 72°C for 25s, 30 cycles; 72°C for 10 minutes.

In the above kit of the present invention, after the sample to be tested is detected by RT-PCR, if the amplified product has a band with a size of 377 bp, then the sample to be tested contains 4/91 infectious bronchitis virus.

The present invention also provides a non-diagnostic method for detecting 4/91 type infectious bronchitis virus, comprising the following steps:

(1) Extracting the total RNA of the sample to be tested, reverse transcription, and obtaining cDNA;

(2) Perform PCR amplification on the cDNA in step (1) using the specific primer pair shown in SEQ ID NO.1-2, and judge whether there is 4/91 infectious bronchitis virus in the sample to be tested according to the amplification result.

Wherein, the target fragment amplified from the positive sample in step (2) is 377bp.

The present invention has the advantages that 1) the amplified target fragment is located in the hypervariable region of the S1 protein of 4/91 type IBV, has a length of 377bp, has good sensitivity and is easy to operate; 2) the method is effective for other common poultry disease pathogens, such as The detection results of avian influenza virus, Newcastle disease virus, infectious bursal virus, avian reovirus, avian adenovirus and avian infectious laryngotracheitis virus were all negative and there was no cross-reaction, indicating that the method also has good Specificity; 3) The inventive method is suitable for carrying out the detection of 4/91 type IBV in poultry production, can not amplify common vaccine strain (H120, LDT3 etc.) and main epidemic strain (QX-like, TW-like etc.) , can only amplify type 4/91 strains, has the characteristics of high specificity, high sensitivity, high efficiency, and low cost, and can quickly differentially diagnose clinical disease materials within 6 hours, overcoming the time-consuming traditional detection methods Shortcomings. This method is suitable for the detection and analysis of a large number of clinical samples, and provides a reliable technical means for the early and rapid diagnosis, monitoring and molecular epidemiological investigation of 4/91 type IBV.

Description of drawings

Fig. 1 is the electrophoresis result of the optimal amplification primer screening. The spotting sequence is M: Marker II; 1: Primer 1 (377bp); 2: Primer 2 (401bp); 3: Primer 3 (518bp).

Figure 2 is the result of primer 2 specificity test. The spotting sequence was M: MarkerII; 1: 4/91 strain; 2: JS strain; 3: GD strain; 4: H52 strain; 5: conn strain; 6: T strain.

Figure 3 is the result of primer 3 specificity test. The spotting sequence was M: MarkerII; 1: 4/91 strain; 2: JS strain; 3: GD strain; 4: H52 strain; 5: conn strain; 6: T strain.

Fig. 4 is the electrophoresis result of the optimal annealing temperature screening for RT-PCR reaction. The spotting sequence is M: MarkerII; 1: 55°C; 2: 57°C; 3: 58°C; 4: 59°C; 5: 61°C.

Fig. 5 is the result of screening electrophoresis for optimal cycle number of RT-PCR reaction. M: Marker II; 1: 28 cycles; 2: 29 cycles; 3: 30 cycles; 4: 31 cycles; 5: 32 cycles; 6: 33 cycles.

Fig. 6 is a genetic phylogenetic tree drawn according to the IBV S1 gene, in which ● shows representative IBV strains of different genotypes detected by the method of the present invention.

Fig. 7 is the electrophoresis result of detecting different genotypes of infectious bronchitis virus selected. The order of adding samples is M: DNA Maker II; P: positive control (4/91 type IBV); N: negative control; 1: T strain (kidney type standard strain); 2: M41 (M type standard strain); 3: YN strain (YN-like); 4: JS strain (QX-like); 5: GD strain (TW-like).

Fig. 8 is the electrophoresis result of detecting other common poultry disease pathogens. Among them, M: DNA Maker II; P: positive control (4/91 type IBV); N: negative control; 1: H5 subtype avian influenza virus; 2: H7 subtype avian influenza virus; 3: H9 subtype avian influenza virus ;4: Newcastle disease virus (NDV); 5: Infectious bursal virus (IBDV); 6: Avian reovirus (REOV); 7: Avian adenovirus (FAdV); 8: Avian infectious laryngotracheitis virus (ILTV).

Figure 9 shows the results of primer specificity detection published in the literature, sample sequence: M: Marker II; P: positive control; N: negative control; 1: T strain; 2: conn strain; 3: H52; 4: JS strain; 5 : GD strain.

detailed description

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

For routine experimental procedures in the following examples, see Molecular Cloning by Sambrook et al. The use of the instrument refers to the operating instructions of the instrument. The LEGEND MICRO 17R low-temperature desktop centrifuge is a product of Thermo Company; the VS-1 vortex oscillator was purchased from Dinghaoyuan Technology Co., Ltd.; the TL-2010S tissue grinding oscillator was purchased from Dinghaoyuan Technology Co., Ltd. Reverse Transcriptase (Reverse Transcriptase) 200U/μl (Promega), Nuclease Inhibitor (Rnase Inhibitor) 50U/μl (Takara), 5 times the volume of reaction buffer (5×Reaction Buffer), dNTP mixture 2.5mM and random Primer (Random Primer) 500 μg/ml (Promega) was purchased from Beijing Appreciation Technology Co., Ltd.; DEPC treated water was purchased from Beijing Minghao Zhiyuan Co., Ltd. Marker II DNA Ladder was purchased from Zhongke Ruitai (Beijing) Biotechnology Co., Ltd. The Veriti 96-Well Thermal Cycler PCR amplification instrument was a product of Applied Biosystems and was purchased from Beijing Chengmao Xingye Technology Development Co., Ltd.; the MINI-Smart small desktop centrifuge was a product of HERO; the HW·SYII-KP3 electric heating constant temperature water tank was purchased from Beijing Changfeng Instrument Co., Ltd.

The biological material selected in the embodiment of the present invention is as follows: avian influenza virus, Newcastle disease virus, infectious bursal virus, avian reovirus, avian adenovirus and avian infectious laryngotracheitis virus, infectious bronchitis virus 4 /91 type, T strain (kidney type standard strain), M41 (M type standard strain), YN strain (YN-like), JS strain (QX-like), GD strain (TW-like), H52 strain (M type Standard strain) and conn strain (M-type standard strain) were preserved and provided by the School of Veterinary Medicine, China Agricultural University.

Embodiment 1 is used to detect the design of the specific primer of 4/91 type infectious bronchitis virus

Specific primers were designed and screened with reference to the hypervariable region of the 4/91 type IBV-S1 gene sequence (GenBank accession number KF377577) registered in GenBank. Among the numerous candidate primers, after repeated screening and comparison tests to exclude possible non-specific matches between the primers and other species sequences, after repeated screening and verification, three sets of candidate primer pairs were finally obtained, and the three sets of Primer pairs were tested and the optimal primer pair was selected. Primers 1, 2, and 3 in Table 1 are all primers for the same target gene, and the positions of the three are adjacent. The electrophoresis results of each primer amplification are shown in Figures 1, 2 and 3. The results show that primers 2 and 3 can amplify other types of IBV strains with poor specificity. Figure 2 shows that primer 2 can amplify JS strain, GD strain, conn strain, and T strain, and Figure 3 shows that it can amplify The target bands of H52 strain and conn strain have poor specificity, so primers 2 and 3 were discarded. Primer 1 amplified the target band with the brightest and no non-specific bands, so primer 1 was selected as the best primer. The upstream primer was between 20500nt-20520nt with GenBank accession number KF377577, and the downstream primer was between 20858nt-20876nt.

Table 1 Alternative primer sequences for detecting 4/91 type IBV

The preferred specific primer pair provided by the present invention is the first group of primer pairs in Table 1, and its sequence is: the upstream primer 5'-TCTGATTGCACTGCTGGTACT-3' (SEQ ID NO.1); and the downstream primer 5'- GCACCCGTAACGTGAGTTG-3' (SEQ ID NO. 2).

Example 2 Detecting RT-PCR Detection Method of 4/91 Type Infectious Bronchitis Virus The Exploration of Optimum Annealing Temperature

1. Pretreatment of test samples

(1) Tissue sample processing: take 100 mg of organ tissue samples, add 0.5 ml of sterilized saline, grind and suspend with a grinder, centrifuge the tissue suspension at 3000 rpm for 30 min, and take the supernatant for detection and analysis.

(2) Allantoic fluid sample processing: after the sample was centrifuged at 3000 rpm for 30 min, the supernatant was taken for detection and analysis.

2. Extraction of total RNA from samples

Extraction was performed according to the instructions of Trizol RNA Extraction Kit (Invitrogen).

3. Reverse transcription into cDNA

Add the following components into a 0.2ml centrifuge tube: 4 μl of RNA solution, 1 μl of random primers, mix gently, bathe in 70°C water for 5 minutes, and ice-bath for 2 minutes, then add the following components in sequence: 4 μl of 5× reaction buffer, 2 μl of dNTP mixture, nuclease Inhibitor 1 μl, reverse transcriptase 0.5 μl, DEPC-treated water 7.5 μl, mix gently, and act at 37°C for 1 hour to obtain sample cDNA.

4. PCR detection

PCR was performed using the primer pair finally determined in Example 1 (the first set of primers).

Different annealing temperature gradients (55°C, 56°C, 57°C, 58°C and 59°C) were designed to perform RT-PCR on type 4/91 IBV.

Add the following ingredients to a 0.2ml centrifuge tube:

After mixing gently, the following reactions were carried out at different annealing temperatures: 94°C for 5 minutes, 94°C for 45s, (55°C, 57°C, 58°C, 59°C and 61°C) for 45s, 72°C for 25s, and 30°C for 30 seconds. cycle, and at the end of the cycle, extend at 72°C for 10 min.

After the PCR reaction, prepare 1.2% agarose gel with 1×TAE electrophoresis buffer and mix the fluorescent dye Gelsafe according to the reference ratio, take 7 μl of PCR product and add it to the gel well, and select an appropriate voltage (4V/cm-10V/cm ) for electrophoresis, and the electrophoresis time is 25-35min. After the electrophoresis, place the gel block on a UV gel imager to observe and take pictures, and determine whether the target band can be amplified in the sample according to the electrophoresis result. A band length of 377bp proves that the sample to be tested is positive for 4/91 type IBV, otherwise it is negative.

The electrophoresis results are shown in Figure 4. 58°C is the brightest band for the purpose of amplification, and too high annealing temperature is not conducive to the combination of primers and templates, so 58°C is selected as the optimal annealing temperature.

Embodiment 3 Detects the RT-PCR detection method of 4/91 type IBV optimal number of cycles to explore

For the pretreatment of the test sample, the extraction of the total RNA of the sample, and the reverse transcription into cDNA, refer to the corresponding method in Example 2. Using the conditions determined in Examples 1 and 2, 6 different cycle numbers (28, 29, 30, 31, 32 and 33) were designed to perform RT-PCR on 4/91 type IBV.

In the 0.2ml centrifuge tube, add composition with embodiment 2. After mixing gently, carry out the following reaction: pre-denaturation at 94°C for 5 minutes, 45s at 94°C, 45s at 58°C, and 25s at 72°C. ℃ extension 10min. For the PCR product electrophoresis experiment, see Example 2. The electrophoresis results are shown in Figure 5. The target band amplified with 30 cycles is similar in brightness to more cycles, and there are no non-specific bands, which can save time and improve detection efficiency. , so choose it as the optimal number of cycles.

Example 4 Establishment of RT-PCR detection method for detecting 4/91 type infectious bronchitis virus

For the pretreatment of the test sample, the extraction of the total RNA of the sample, and the reverse transcription into cDNA, refer to the corresponding method in Example 2.

Add in a 0.2ml centrifuge tube, see Example 2 for the ingredients.

After mixing gently, use the following reaction program: initial denaturation at 94°C for 5 min, 30 cycles (94°C for 45 s, 58°C for 45 s, 72°C for 25 s), and finally extension at 72°C for 10 min. After the PCR reaction, use 1× Prepare 1.2% agarose gel with TAE electrophoresis buffer and mix the fluorescent dye Gelsafe according to the reference ratio, take 7 μl of PCR product and add it to the gel well, select the appropriate voltage (4V/cm-10V/cm) for electrophoresis, and the electrophoresis time is 25 -35min. After the electrophoresis, put the gel block on a UV gel imager to observe and take pictures. Determine whether the target band can be amplified in the sample according to the electrophoresis result. If the target band is 377bp in length, it proves to be The test sample is positive for 4/91 type IBV, otherwise it is negative.

Embodiment 5 Detects the characteristic evaluation of the RT-PCR detection method of 4/91 type infectious bronchitis virus

1. Specific detection of different genotypes of infectious bronchitis virus

Carry out according to the method established in Example 4.

Utilize above-mentioned method to detect different genotype IBV (as shown in Figure 6) respectively, electrophoresis result is as shown in Figure 7, the result illustrates, the method that the embodiment of the present invention 4 establishes can only detect 4/91 type IBV but detects not Other serotypes of IBV can be detected, so the IBV genotype can be identified on the test samples, which shows that the method has good sensitivity and specificity. 2. Specific detection of pathogens of common poultry diseases

Utilize embodiment 4 to establish method to other common poultry disease pathogens, avian influenza virus, Newcastle disease virus, infectious bursal virus, avian reovirus, avian adenovirus and avian infectious laryngotracheitis virus are detected, the result As shown in Figure 8, the results show that, except for the positive control, the other test objects are all negative, indicating that the method established in Example 4 has good specificity for 4/91 type IBV in the process of detecting other common pathogens in poultry. sex.

3. Compared with the existing technology

The prior art records the establishment and application of chicken infectious bronchitis virus 793B RT-PCR detection method (Yu Shenye, Diao Youxiang, etc., Chinese Journal of Veterinary Medicine, July 2007). The primer sequences used are as follows: upstream ACGAATTCATGTTGGGCAAACCGCT; downstream ATGTCGACAAGTATCAACGCCACC, Chicken Infectious Bronchitis Virus 793B is also IBV 4/91 type. The applicant used the above primers to carry out specificity detection experiments, and found that the above primers can detect IBV T strain (kidney type standard strain) in addition to detecting IBV 4/91 type. It can be seen that the specificity of the above primers is not as specific as the primers of the present invention. powerful. The results are shown in Figure 9.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. it is a kind of for detect 4/91 type IBV specific primer pair, it is characterised in that the primer To target gene for IBV S1 genes hypervariable region.

2. it is a kind of for detect 4/91 type IBV specific primer pair, it is characterised in that its nucleotides Sequence contains the sequence as shown in SEQ ID NO.1-2.

3. specific primer described in claim 1 or 2 is in discriminating IBV serotype kit is prepared Application.

4. specific primer described in claim 1 or 2 is in 4/91 type IBV kit of detection is prepared Application.

5. containing the detection reagent of specific primer pair described in claim 1 or 2.

6. containing the kit of specific primer pair described in claim 1 or 2.

7. kit as claimed in claim 6, it is characterised in that its working procedure is：94℃5min；94 DEG C of 45s, 58 DEG C 45s, 72 DEG C of 25s, 30 circulations；72℃10min.

8. kit as claimed in claims 6 or 7, it is characterised in that carry out to sample to be tested after RT-PCR detections, amplification is produced If thing has the band of 377bp sizes, 4/91 type IBV is contained in sample to be tested.

9. it is a kind of detection 4/91 type IBV non-diagnostic purpose method, it is characterised in that including following step Suddenly：

(1) total serum IgE of sample to be tested is extracted, reverse transcription obtains cDNA；

(2) performing PCR amplification is entered to the cDNA to step (1) using specific primer described in claim 1 or 2, according to amplification knot Fruit judges whether there is 4/91 type IBV in sample to be tested.

10. method as claimed in claim 9, it is characterised in that the purpose fragment of the positives sample amplification of step (2) is 377bp。