CN111424113A

CN111424113A - Primer group, kit and method for detecting chicken respiratory diseases

Info

Publication number: CN111424113A
Application number: CN201911219209.6A
Authority: CN
Inventors: 周祖涛; 杨荣坤; 肖运才; 胡思顺; 李自力; 刘梅; 许青荣; 崔卫涛; 毕丁仁
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-07-17

Abstract

The primer group comprises at least one of a first primer pair, a second primer pair, a third primer pair and a fourth primer pair, wherein the first primer pair comprises a first forward primer and a first reverse primer, the second primer pair comprises a second forward primer and a second reverse primer, the third primer pair comprises a third forward primer and a third reverse primer, and the fourth primer pair comprises a fourth forward primer, a fourth reverse primer and a fourth reverse primer.

Description

Primer group, kit and method for detecting chicken respiratory diseases

Technical Field

The disclosure relates to the technical field of biology, in particular to a primer group, a kit and a method for detecting respiratory diseases of chickens.

Background

The respiratory disease of chicken is high in incidence rate, can infect chickens of different ages of days, is very common and serious disease in recent years, and is very difficult to prevent and treat, wherein, the infectious laryngotracheitis virus (I L TV), the Mycoplasma Synoviae (MS), the Mycoplasma Gallisepticum (MG) and the Avibacterium Paragallinarum (APG) are a plurality of important respiratory disease pathogens which seriously harm the development of the chicken industry in China, the pathogenesis characteristics, clinical symptoms and pathological anatomical change of the respiratory disease of the chicken are similar, the identification is difficult by naked eyes, the clinical mixed infection or secondary infection appears, which brings great difficulty to the identification of the respiratory disease of the chicken.

The traditional detection methods mainly comprise pathogen separation and identification, serological tests and the like, but the methods have the problems of high cost, complex operation, time consumption and the like, and are difficult to quickly identify mixed infection. Currently, with the development of molecular biology, the PCR (Polymerase Chain Reaction) technology has been widely applied to the detection of poultry diseases, and a multiplex PCR detection technology has been established. However, in the detection, a plurality of pairs of primers are required to be amplified competitively at the same time, and the obtained PCR amplification product is required to be analyzed by nucleic acid gel electrophoresis, but the current nucleic acid gel electrophoresis analysis is difficult to distinguish the bands with the size of 50-100 bp, and the lower the corresponding sensitivity is, the more complex primer dimer is easily generated by adding one pair of primers, so that the existing detection can not realize high-sensitivity and high-throughput detection on various pathogens in a real sense.

Disclosure of Invention

In order to solve the problems in the prior art, the embodiment of the disclosure provides a primer group, a kit and a method for detecting chicken respiratory diseases. The technical scheme is as follows:

in one aspect, the present disclosure provides a primer set for detecting respiratory diseases of chickens, the primer set comprising: at least one of a first primer pair, a second primer pair, a third primer pair and a fourth primer pair, and a universal primer pair,

the first primer pair comprises: a first forward primer and a first reverse primer, wherein the sequence of the first forward primer is shown as SEQ ID NO. 1 in the sequence table, the sequence of the first reverse primer is shown as SEQ ID NO. 2 in the sequence table,

the second primer pair comprises: a second forward primer and a second reverse primer, wherein the sequence of the second forward primer is shown as SEQ ID NO. 3 in the sequence table, the sequence of the second reverse primer is shown as SEQ ID NO. 4 in the sequence table,

the third primer pair comprises: a third forward primer and a third reverse primer, wherein the sequence of the third forward primer is shown as SEQ ID NO. 5 in the sequence table, the sequence of the third reverse primer is shown as SEQ ID NO. 6 in the sequence table,

the fourth primer pair comprises: a fourth forward primer and a fourth reverse primer, wherein the sequence of the fourth forward primer is shown as SEQ ID NO. 7 in the sequence table, and the sequence of the fourth reverse primer is shown as SEQ ID NO. 8 in the sequence table;

the universal primer pair comprises: a universal forward primer and a universal reverse primer.

Specifically, the sequence of the universal forward primer is shown as SEQ ID NO. 9 in the sequence table, and the sequence of the universal reverse primer is shown as SEQ ID NO. 10 in the sequence table.

In another aspect, the present disclosure provides a kit for detecting respiratory diseases of chickens, which comprises the above primer set.

Specifically, the final concentrations of the first forward primer and the first reverse primer are both 0.75 mu M/L, the final concentrations of the second forward primer and the second reverse primer are both 1.0 mu M/L, the final concentrations of the third forward primer, the third reverse primer, the fourth forward primer and the fourth reverse primer are all 1.25 mu M/L, and the final concentrations of the forward universal primer and the reverse universal primer are both 20 mu M/L.

Specifically, the kit further comprises: negative control sample, positive control sample, EX Taq polymerase and RNaseFreedH₂O。

Specifically, the final concentration of EX Taq polymerase was 5U/. mu. L.

Specifically, the positive control sample comprises a positive standard plasmid containing a TK gene of the avian infectious laryngotracheitis virus, a positive standard plasmid containing a Mycoplasma gallisepticum 16SrRNA gene, a positive standard plasmid containing a Mycoplasma synoviae V L HA gene and a positive standard plasmid containing an avibacterium paragallinarum HA gene.

Specifically, the negative control sample was pMD18-T unloaded plasmid.

In another aspect, the present disclosure provides a method for detecting respiratory diseases of chickens by using the primer set, which is characterized in that the method comprises:

extracting DNA of a sample to be detected as a template;

carrying out amplification reaction on the template by adopting the primer group to obtain an amplification product;

and analyzing the chicken respiratory diseases by adopting capillary electrophoresis on the amplification products.

Specifically, the amplification reaction comprises a first stage and a second stage, and the amplification procedure of the first stage comprises for each cycle: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s, and extension at 72 ℃ for 30s for 10 cycles;

the second phase of the amplification procedure comprises for each cycle: denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 45s, and extension at 72 ℃ for 45s for 20 cycles; final extension: extension at 72 ℃ for 10 min.

The technical scheme provided by the embodiment of the disclosure HAs the advantages that the primer group provided by the disclosure can be used for efficiently detecting whether a sample to be detected carries the TK gene of the avian infectious laryngotracheitis virus, the Mycoplasma gallisepticum 16SrRNA gene, the Mycoplasma synoviae V L HA gene and the avian paragallinarum HA gene at one time, when the chicken suffers from respiratory diseases, the common primer pair can be frequently caused by common infection of several of the four pathogens, the amplification preference problem in multiplex PCR amplification can be effectively avoided, and the target gene can be efficiently expressed and amplified.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a diagram of capillary electrophoresis analysis provided in the third embodiment of the present disclosure;

FIG. 2 is a diagram of single-plex capillary electrophoresis analysis provided in the third embodiment of the present disclosure;

FIG. 3 is a capillary electrophoresis analysis diagram of MG provided in the third embodiment of the disclosure;

FIG. 4 is a diagram of capillary electrophoresis analysis of MS provided in the third embodiment of the present disclosure;

FIG. 5 is a diagram of capillary electrophoresis analysis of APG provided in example III of the present disclosure;

FIG. 6 is a diagram of capillary electrophoresis analysis for simulating mixed infection of MG and MS provided in the third embodiment of the disclosure;

FIG. 7 is a diagram of capillary electrophoresis analysis of simulated I L T, MS and APG mixed infection provided in example III of the present disclosure.

Detailed Description

To make the objects, technical solutions and advantages of the present disclosure more apparent, embodiments of the present disclosure will be described in detail with reference to the accompanying drawings.

The experimental methods used in the examples of the present disclosure are all routine experimental methods unless otherwise specified.

Reagents and instruments used in the examples of the present disclosure and the like are commercially available in general unless otherwise specified.

Example one

The embodiment of the disclosure provides a primer group for detecting chicken respiratory diseases, which comprises: at least one of a first primer pair, a second primer pair, a third primer pair, and a fourth primer pair, and a universal primer pair, the first primer pair comprising: a first forward primer and a first reverse primer, wherein the sequence of the first forward primer is shown as SEQ ID NO. 1 in the sequence table, the sequence of the first reverse primer is shown as SEQ ID NO. 2 in the sequence table, and the second primer pair comprises: a second forward primer and a second reverse primer, wherein the sequence of the second forward primer is shown as SEQ ID NO. 3 in the sequence table, the sequence of the second reverse primer is shown as SEQ ID NO. 4 in the sequence table, and the third primer pair comprises: a third forward primer and a third reverse primer, wherein the sequence of the third forward primer is shown as SEQ ID NO. 5 in the sequence table, the sequence of the third reverse primer is shown as SEQ ID NO. 6 in the sequence table, and the fourth primer pair comprises: a fourth forward primer and a fourth reverse primer, wherein the sequence of the fourth forward primer is shown as SEQ ID NO. 7 in the sequence table, and the sequence of the fourth reverse primer is shown as SEQ ID NO. 8 in the sequence table.

The universal primer pair comprises: the sequence of the universal forward primer is shown as SEQ ID NO. 9 in the sequence table, and the sequence of the universal reverse primer is shown as SEQ ID NO. 10 in the sequence table.

Specifically, the length of an amplification product (namely the target peak position) is estimated to be 184bp by taking a TK gene of the avian infectious laryngotracheitis virus (I L TV) as a first primer pair of a target gene, the target sequence is Genbank: S83714.1, the length of the amplification product (namely the target peak position) is estimated to be 200bp by taking a Mycoplasma Gallisepticum (MG)16SrRNA gene as a second primer pair of the target gene, the target sequence is Genbank: JN935873.1, the length of the amplification product (namely the target peak position) is estimated to be 138bp by taking a Mycoplasma Synoviae (MS) V L HA gene as a third primer pair of the target gene, the target sequence is Genbank: GU084382.1, the length of the amplification product (namely the target peak position) is estimated to be 160bp by taking an Avian Paragallibacterium Gallinarum (APG) HA gene as a fourth primer pair of the target gene, and the target sequence is Genbank: KJ 621068.1.

The primer group provided by the disclosure can be used for efficiently detecting whether a sample to be detected carries the TK gene, the Mycoplasma gallisepticum 16SrRNA gene, the Mycoplasma synoviae V L HA gene and the avian paragallibacterium HA gene at one time, and when the chicken suffers from respiratory diseases, the chicken is often caused by common infection of several of the four pathogens.

Example two

The disclosed embodiment provides a kit for detecting chicken respiratory diseases, which comprises a primer group provided by the disclosed embodiment, and the kit further comprises a universal forward primer with a sequence shown as SEQ ID NO. 9 in a sequence table and a universal reverse primer with a sequence shown as SEQ ID NO. 10 in the sequence table.

Furthermore, the positive control sample comprises a positive standard plasmid containing a TK gene of the avian infectious laryngotracheitis virus, a positive standard plasmid containing a Mycoplasma gallisepticum 16SrRNA gene, a positive standard plasmid containing a Mycoplasma synoviae V L HA gene and a positive standard plasmid containing an avian paragallibacterium HA gene.

Further, the negative control sample was pMD18-T unloaded plasmid.

Furthermore, the concentration of the first forward primer and the first reverse primer is 0.75 μ M/L, the concentration of the second forward primer and the second reverse primer is 1.0 μ M/L, the concentration of the third forward primer, the concentration of the third reverse primer, the concentration of the fourth forward primer and the concentration of the fourth reverse primer are 1.25 μ M/L, and the concentration of the universal forward primer and the concentration of the universal reverse primer are 20 μ M/L.

The primer group in the kit provided by the disclosure can be used for detecting whether a sample to be detected carries the TK gene, the Mycoplasma gallisepticum 16SrRNA gene, the Mycoplasma synoviae V L HA gene and the avibacterium paragallinarum HA gene at one time and efficiently, when the chicken suffers from respiratory diseases, the chicken often suffers from common infection of several of the four pathogens, the kit can be used for simultaneously detecting whether the sample to be detected suffers from the respiratory diseases of the chicken caused by the four pathogens, so that the diseased condition of the sample to be detected can be comprehensively obtained, and meanwhile, the primer group HAs higher specificity, sensitivity and accuracy.

EXAMPLE III

The embodiment of the invention provides a method for detecting chicken respiratory diseases by adopting a primer group provided by the first embodiment of the disclosure, which comprises the following steps:

extracting DNA of Mycoplasma Gallisepticum (MG), Mycoplasma Synoviae (MS), Avian Paragallinarum (APG) and avian infectious laryngotracheitis virus (I L TV) respectively, and mixing to obtain a template;

carrying out amplification reaction on a template by using the primer group provided by the first embodiment of the disclosure to obtain an amplification product;

the amplification products were analyzed by capillary electrophoresis.

In this example, a commercial DNA/RNA extraction kit (purchased from Beijing Olympic Biotechnology Ltd.) was used to extract DNA from chicken embryo fluid containing infectious laryngotracheitis virus (Hubei strain), mycoplasma gallisepticum (F/S6/HS strain), mycoplasma synoviae (1853 strain), and avian paragallibacterium (Hubei strain) isolated and identified and preserved in the animal medical college of Huazhong university of agriculture and immunology laboratory according to the instruction of the DNA/RNA extraction kit, and meanwhile, pMD18-T plasmid containing infectious laryngotracheitis virus TK gene at a final concentration of 1ng/μ L, Mycoplasma gallisepticum 16SrRNA gene at a final concentration of 1ng/μ L, mixed avian paragallisepticum HA gene at a final concentration of 1ng/μ L were used as negative controls, and L HA gene containing infectious laryngotracheitis virus, and 1ng/μ L positive plasmid mixed avian paragallisepticum HA gene at a final concentration of 1ng/μ L were used as standard controls.

The four DNAs were mixed and used as templates, and the final concentration was 1 ng/. mu. L as a template for multiplex PCR reactions, respectively, amplification was carried out on a Thermo PCR amplification apparatus using a 30. mu. L amplification reaction system, and the PCR reactions were repeated three times at different times and with different PCR apparatuses.

In this example, the final concentrations of the first forward primer and the first reverse primer were 0.75. mu.M/L, the final concentrations of the second forward primer and the second reverse primer were 1.0. mu.M/L, the final concentrations of the third forward primer, the third reverse primer, the fourth forward primer and the fourth reverse primer were 1.25. mu.M/L, and the final concentrations of the forward universal primer and the reverse universal primer were 20. mu.M/L.

The amplification reaction system comprises a template 2 mu L, a universal forward primer and a universal reverse primer which are respectively 0.6 mu L, and mixed primers of a first forward primer, a second forward primer, a third forward primer, a fourth forward primer and a first reverse primer, a second reverse primer, a third reverse primer and a fourth reverse primer are respectively 0.6 mu L and 5U/. mu. L of EX Taq polymerase 15 mu L FreeDH₂O9.4. mu. L, and the total reaction volume is 30. mu. L.

The amplification reaction comprises a first stage and a second stage, the amplification procedure of the first stage comprising for each cycle: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 45s, and extension at 72 ℃ for 30s for a total of 10 cycles. The second phase of the amplification procedure comprises for each cycle: denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 45s, and extension at 72 ℃ for 45s, for a total of 20 cycles, final extension: extending for 10min at 72 ℃, and storing at 4 ℃. Obtaining PCR amplification products.

The specificity of the primer group is detected by adopting capillary electrophoresis, which comprises the following steps:

qsep Using a fully automated nucleic acid analysis System₁₀₀ ^TMDetecting each PCR amplification product, wherein the specific operation steps are as follows:

(1)Qsep₁₀₀ ^TMa full-automatic nucleic acid protein analysis system is characterized in that 2/3 volumes of distilled water are placed in positions of cleaning tanks P (park), W (Wash) and C (clean), 2/3 volumes of Separation Buffer is placed in a position S, 20bp-1000bp of Alignment Marker is placed in a MA1 hole, and corresponding Size Marker (C109200-100) is placed in a MA2 hole (note that liquid level heights in P, W, C and S tanks are proper at the positions of slow scale lines and cannot be lower than 2/3 of the tank volume); mu.l of Alignment Marker and Sizemarker were added to the PCR tube (removing the tube cap) and covered with 30. mu.l of Mineral Oil (Mineral Oil) to prevent evaporation. Click on Change Sample (A) and put the Sample; and placing the card in an SI card holder, and selecting 6KV voltage to carry out high-voltage glue passing inspection (HV check) and card holder correction.

(2) Directly placing the amplification product into a Sample position, and inputting Sample information (Sample ID) at a corresponding position; the program selects an Alignment Marker of 20bp-1000bp and a Method of 6KV (note: the electrophoresis voltage of the selected Method is consistent with the voltage when the card clip is corrected); sample Duration 10S; run (detection repetition number) 1 times and Separation Duration 300S. After the sample detection is finished, the user-defined Qsep is used₁₀₀ ^TMAnd the full-automatic nucleic acid protein analysis system analyzes the separated fragments and judges the result.

As shown in FIG. 1, it can be seen from FIG. 1 that all the multiplex PCR results using the extracted genomic DNA of I L TV, MG, MS, and APG sequences as templates show positive results, the capillary electrophoresis and fragment analysis sizes shown in FIG. 1 respectively show that MS is 145bp, APG is 165bp, I L TV is 189bp, and MG is 208bp, and the capillary electrophoresis matched clip has an error of 1-7 bp, so the sizes of the amplification products are consistent with the size of the target fragment, and the target gene sequence is identified by sequencing.

Single-primer mixed template primer specificity verification

The final concentrations of the first forward primer and the first reverse primer are both 0.75 mu M/L, the final concentrations of the second forward primer and the second reverse primer are both 1.0 mu M/L, the final concentrations of the third forward primer, the third reverse primer, the fourth forward primer and the fourth reverse primer are both 1.25 mu M/L, the final concentrations of the forward universal primer and the reverse universal primer are both 20 mu M/L, the DNAs of the avian infectious laryngotracheitis virus (I L TV), the Mycoplasma Gallisepticum (MG), the Mycoplasma Synoviae (MS) and the Avian Paragallinarum (APG) obtained from allantoic fluid are used as templates, the primers are respectively used for carrying out single PCR amplification, the actual detection size of a target gene is determined, a 30 mu L amplification reaction system is adopted for carrying out amplification on a Thermo PCR amplification instrument, and each DNA is subjected to 3 times of repeated operations.

The amplification reaction system comprises a template 1 mu L, a universal forward primer and a universal reverse primer which are respectively 1 mu L, and the first forward primer or the second forward primer or the third forward primer or the fourth forward primer and the first reverse primer or the second reverse primer or the third reverse primer or the fourth reverse primer are respectively 0.5 mu L and 5U/mu L of EX Taq polymerase 15 mu L Free dH₂O11. mu. L, total 30. mu. L.

Capillary electrophoresis

The same automatic nucleic acid analysis System Qsep as in example 3 was used₁₀₀ ^TMThe detection results of the PCR products are shown in FIGS. 2 to 5, and it can be seen from FIG. 2 that the sizes of the capillary electrophoresis and the fragment analysis are respectively 187bp for I L TV, 205bp for MG, 142bp for MS, and 163bp for APG, respectively, for FIG. 3 and FIG. 4, because of the capillary electrophoresisThe electrophoresis matched clamp has an error of 1-7 bp, so the size of the amplification product conforms to the size of a target fragment, and non-specific amplification does not exist, which indicates that the primer pair designed in the experiment has high specificity.

The sensitivity of the primer group is detected, and the specific steps are as follows:

and (3) identifying the sensitivity of single PCR capillary electrophoresis:

DNA of the infectious laryngotracheitis virus (I L TV), Mycoplasma Gallisepticum (MG), Mycoplasma Synoviae (MS) and Avibacterium Paragallinarum (APG) obtained from allantoic fluid is used as a template, full-length amplification primers of the TK gene of I L TV, the 16SrRNA gene of MG, the V L HA gene of MS and the HA gene of APG are used for PCR amplification, PCR amplification products are subjected to electrophoresis detection by 2% agarose gel, correct strips are cut and recovered by using a gel recovery kit (the gel recovery refers to DNA gel recovery kit test instruction of Beijing all-type gold biotechnology Limited company) and purified, the purified PCR amplification products are connected with a pMD18-T vector to obtain a connection product, the connection product is transformed into a competent cell E.coli DH5 α for culture, the PCR-identified positive clone product as a primary recombinant plasmid (pMD18-T-A, pMD18-T-B, pMD 18-C, pMD 18-T), the homology of each DNA sequence of the PCR amplification product is compared with a plasmid sequence of a Beijing full-length amplification primer of the DNA of the PcJG, and the DNA sequence of the DNA obtained by using a PCR amplification kit of the PCR amplification products of the Ministributaceae under the Beijing technology of the order of the NC26 and the NC26, and.

Respectively extracting the 4 recombinant plasmids pMD18-T-A, pMD18-T-B, pMD18-T-C, pMD18-T-D, measuring the concentration of each recombinant plasmid by using a spectrophotometer, calculating the copy number according to the molecular mass and the length of each recombinant plasmid, and sequentially diluting the target gene concentration of each recombinant plasmid to 10⁶copies/μL、10⁵copies/μL、10⁴copies/μL、10³copies/μL、10²copies/μ L and 10¹copies/μL。

Respectively pairing the first primer pair, the second primer pair, the third forward primer pair, the fourth primer pair and the universal primer pairThe single PCR detection and capillary electrophoresis analysis are carried out on the recombinant plasmids pMD18-T-A, pMD18-T-B, pMD18-T-C and pMD18-T-D with the concentration, the sensitivity result of the single PCR shows that the minimum detection amount of I L TV is 10²The lowest detection of copies/μ L was 10²The lowest detection of copies/μ L was 10²The lowest detection of copies/μ L was 10³copies/μ L it can be seen that the first primer pair, the second primer pair, the third forward primer pair, and the fourth primer pair provided by the present disclosure all have higher sensitivity.

And (3) identifying the sensitivity of the multiple PCR capillary electrophoresis:

and mixing a first forward primer, a first reverse primer, a second forward primer, a second reverse primer, a third forward primer, a third reverse primer, a fourth forward primer and a fourth reverse primer in equal proportion to ensure that the working concentration of the primers is 1 mu M/L, mixing all positive standard plasmids in equal proportion as templates, taking pMD18-T no-load plasmids as negative controls, and establishing a multiplex PCR detection reaction system under the same single PCR with the rest reaction conditions.

Reaction System (30. mu. L) four kinds of the above DNA mixed template 2. mu. L, forward universal primer and reverse universal primer each 1.2. mu. L, and mixed primers of first forward primer, second forward primer, third forward primer, fourth forward primer and first reverse primer, second reverse primer, third reverse primer and fourth reverse primer 0.6. mu. L, 5U/. mu. L of EX Taq polymerase 15. mu. L Free dH₂O 9.4μL。

Reaction procedure: each cycle of the specific chimeric primer amplification stage comprises 10 cycles of pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s and extension at 72 ℃; the amplification procedure for the amplification reaction (amplification of the non-amplified forward and reverse universal primers) included for each cycle: denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 45s, extension at 72 ℃ for 45s, for 20 cycles, final extension: extending for 10min at 72 ℃, and storing at 4 ℃.

And (3) identifying the sensitivity of the multiplex primer group:

and carrying out multiplex PCR detection and capillary electrophoresis analysis on the first forward primer, the first reverse primer, the second forward primer, the second reverse primer, the third forward primer, the third reverse primer, the fourth forward primer and the fourth reverse primer which are mixed in equal proportion to the recombinant plasmids pMD18-T-A, pMD18-T-B, pMD18-T-C and pMD18-T-D which are mixed in equal proportion and have each concentration. And (3) directly carrying out capillary electrophoresis analysis on the PCR product, and analyzing the separated fragments by using the self-defined fragment analysis parameters after the capillary electrophoresis is finished so as to determine the actual detection length corresponding to each target gene.

The result shows that the minimum detection amount of I L TV in the multiplex PCR reaction system is 10²The lowest detection of copies/μ L was 10³The lowest detection of copies/μ L was 10³The lowest detection of copies/μ L was 10³copies/mu L, the minimal detection limit of the primer group is 10 when 4 pathogenic microorganisms exist simultaneously³copies/μL。

Detection of the accuracy of the primer set

A multiplex PCR detection simulating clinical mixed infection is carried out by taking clinical samples (clinical samples are from clinic samples of dead chicken in different areas in Hubei, stored and provided by Wuhan Guanyu Biotechnology Limited, wherein the clinic samples comprise mixed tissue disease material samples of cotton swabs of chicken throats, throats of dead chicken, trachea, livers, kidneys and the like, the samples are from laying hens, broilers, breeding hens and chicks in different days of age, and identifying the samples as pathogens of Mycoplasma Synovialis (MS), Mycoplasma Gallisepticum (MG), Avian Paragallibacterium Gallisepticum (APG) and infectious laryngotracheitis virus (I L TV) through conventional laboratory methods such as pathogen separation, sequencing identification and the like, extracting sample DNA according to the method, selecting 2-3 random combinations from the DNAs of the 4 pathogens, dividing the combinations into two groups of tests, wherein the first group is that the MG and MS are mixed to be used as a template, the second group is that the MG and MS are mixed to be used as a template, and an amplification reaction system and an amplification reaction program are the same as the amplification reaction program.

The results are shown in fig. 6 and fig. 7 respectively, in fig. 6, the first group detects two target peaks of 165bp and 188bp without other miscellaneous peaks, which indicates that the sample to be detected contains DNA of two pathogens of MG and MS and is consistent with the reality, and in fig. 7, the second group detects three target peaks of 142bp, 165bp and 208bp without other miscellaneous peaks, which indicates that the sample contains DNA of three pathogens of I L TV, MS and APG and is consistent with the reality, so that the primer group, the kit and the detection method provided by the present disclosure have high accuracy.

The detection method provided by the disclosure combines multiple PCR amplification with capillary electrophoresis, utilizes the characteristics of double primers, adopts two-step amplification, effectively avoids the preference problem in the conventional multiple PCR amplification, and improves the amplification efficiency; the method is combined with capillary electrophoresis, is more environment-friendly and safer than common gel electrophoresis, avoids the use of toxic and harmful nucleic acid dye, improves the resolution ratio, can distinguish target fragments with a difference of more than 7bp, and improves the detection accuracy. In addition, after the universal primer pair is added into the detection method provided by the disclosure, the competition among the primer pairs in the multiplex PCR amplification is reduced, the target gene is efficiently amplified, and the concentration of the primer group in an amplification system is reduced, so that the use amount of the reagent is saved, and the detection cost is reduced.

The above description is only exemplary of the present disclosure and is not intended to limit the present disclosure, which is to be construed in any way as imposing limitations thereon, such as the appended claims, and all changes and equivalents that fall within the true spirit and scope of the present disclosure.

Sequence listing

<110> university of agriculture in Huazhong

<120> primer group, kit and method for detecting chicken respiratory diseases

<160>10

<170>SIPOSequenceListing 1.0

<210>1

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

aggtgacact atagaatatg ccccactaag aataatg 37

<210>2

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

gtacgactca ctatagggat gcaacaggga aacacaa 37

<210>3

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

aggtgacact atagaatatt gaaagtgtgg ggagcaa 37

<210>4

<211>38

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

gtacgactca ctatagggac gtcaattccg tttgagtt 38

<210>5

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

aggtgacact atagaatatg agacgggttt atgccta 37

<210>6

<211>39

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

gtacgactca ctatagggag cttctctaaa tcaggcttt 39

<210>7

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

aggtgacact atagaatagt tggaaaacat acaaatcacg 40

<210>8

<211>41

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gtacgactca ctatagggaa gttggtttat aatcagaacg a 41

<210>9

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

aggtgacact atagaata 18

<210>10

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gtacgactca ctataggga 19

Claims

1. A primer group for detecting chicken respiratory diseases is characterized by comprising: at least one of a first primer pair, a second primer pair, a third primer pair and a fourth primer pair, and a universal primer pair,

2. The primer group of claim 1, wherein the sequence of the universal forward primer is shown as SEQ ID NO. 9 in the sequence Listing, and the sequence of the universal reverse primer is shown as SEQ ID NO. 10 in the sequence Listing.

3. A kit for detecting respiratory diseases of chickens, which comprises the primer set of claim 1.

4. The kit of claim 3, wherein the final concentration of the first forward primer and the first reverse primer is 0.75 μ M/L, the final concentration of the second forward primer and the second reverse primer is 1.0 μ M/L, the final concentration of the third forward primer, the third reverse primer, the fourth forward primer and the fourth reverse primer is 1.25 μ M/L, and the final concentration of the forward universal primer and the reverse universal primer is 20 μ M/L.

5. The kit of claim 3, further comprising: positive control sample, negative control sample, EX Taq polymerase and RNase Free dH₂O。

6. The kit of claim 5, wherein EX Taq polymerase is present at a final concentration of 5U/μ L.

7. The kit of claim 5, wherein the positive control sample comprises a positive standard plasmid containing TK gene of the avian infectious laryngotracheitis virus, a positive standard plasmid containing 16SrRNA gene of mycoplasma gallisepticum, a positive standard plasmid containing V L HA gene of mycoplasma synoviae and a positive standard plasmid containing HA gene of avibacterium paragallinarum.

8. The kit of claim 5, wherein the negative control sample is pMD18-T unloaded plasmid.

9. A method for detecting respiratory diseases of chickens using the primer set of claim 1, comprising:

extracting DNA of a sample to be detected as a template;

10. The method of claim 9, wherein the amplification reaction comprises a first stage and a second stage, and wherein the first stage of the amplification procedure comprises, for each cycle: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 45s, and extension at 72 ℃ for 30s for 10 cycles;