CN-121975978-A - Combined detection method for avian immunosuppressive diseases based on multiplex fluorescence quantitative PCR

CN121975978ACN 121975978 ACN121975978 ACN 121975978ACN-121975978-A

Abstract

The invention discloses a combined detection method for fowl immune inhibitory diseases based on multiple fluorescence quantitative PCR, which comprises the steps of sequentially determining detection targets, designing specific primers and probes, preparing positive standard, preprocessing samples, extracting nucleic acid, purifying nucleic acid samples, establishing a multiple fluorescence quantitative PCR reaction system, setting multiple fluorescence quantitative PCR reaction programs, amplifying, analyzing and judging results; based on the method, the multiple fluorescent quantitative PCR detection system and the specific primer probe are designed, so that the method has the advantages of realizing synchronous detection of multiple avian immunosuppressive pathogens, needing no multiple independent detection on the same sample, simplifying operation flow, reducing detection cost and reducing sample waste, and solves the problems of low efficiency and high cost of the traditional single pathogen detection method.

Inventors

LIU XIAOLIANG
DU XUSHENG
ZHANG QIANQIAN

Assignees

杭州乐毅生物科技有限公司

Dates

Publication Date: 20260505
Application Date: 20260126

Claims (10)

1. The combined detection method for the avian immunosuppressive diseases based on the multiplex fluorescence quantitative PCR is characterized by comprising the following specific steps of: s1, determining a detection target and designing specific primers and probes: S11, according to the genome sequence of the common avian immunosuppressive disease pathogen, performing sequence comparison analysis by using bioinformatics software, and screening gene fragments in each pathogen genome to serve as detection targets; s12, designing specific primers and fluorescent probes based on the screened pathogenic target gene sequences; S2, preparing a positive standard substance: s21, respectively amplifying all the pathogen target gene fragments determined in the S1 by adopting a PCR method, respectively cloning all amplified target gene fragments into a pMD18-T vector, and constructing a recombinant plasmid; s22, sequencing and verifying recombinant plasmids, and selecting the recombinant plasmids with correct sequencing as positive cloning plasmids; s23, respectively carrying out gradient dilution on each positive cloning plasmid to obtain a gradient positive standard; s3, sample pretreatment and nucleic acid extraction: S31, collecting a tissue sample or a blood sample of poultry to be detected, washing the tissue sample with sterile physiological saline, shearing and crushing the tissue sample, adding liquid nitrogen, grinding the crushed tissue sample or the blood sample into powder, and extracting total nucleic acid in the sample by using a virus nucleic acid extraction kit; S32, detecting the concentration and purity of the extracted nucleic acid by using a nucleic acid concentration detector after the extraction is completed; s4, purifying a nucleic acid sample: S41, purifying the nucleic acid sample extracted in the step S3 to remove PCR inhibitors; S42, detecting inhibitor residues of the sample by using a fluorescent quantitative PCR inhibitor detection kit after purification is completed, and detecting the concentration and purity of the purified nucleic acid by using a nucleic acid concentration detector again; s5, establishing a multiple fluorescence quantitative PCR reaction system based on the nucleic acid sample optimized in the S4 as a template, combining the specific primer and the probe designed in the S1 as an amplification primer and a detection probe, and taking the positive standard prepared in the S2 as a reference to establish the multiple fluorescence quantitative PCR reaction system; s6, setting a multiplex fluorescence quantitative PCR reaction program and amplifying: S61, placing the reaction system prepared in the step S5 into a fluorescence quantitative PCR instrument, and setting a reaction program; s62, setting positive control, negative control and blank control at the same time; S7, result analysis and judgment: s71, after the PCR amplification is finished, analyzing the amplification curve and the melting curve by using analysis software of a fluorescence quantitative PCR instrument.
2. The combined detection method for avian immunosuppressive diseases based on multiplex fluorescence quantitative PCR according to claim 1, wherein the common avian immunosuppressive disease pathogens in S11 comprise infectious bursal disease virus, marek 'S disease virus, reticuloendotheliosis virus, avian leukemia virus and infectious anaemia virus, wherein the infectious bursal disease virus targets VP2 gene, marek' S disease virus targets meq gene, the reticuloendotheliosis virus targets env gene, the avian leukemia virus targets gp85 gene and the infectious anaemia virus targets VP1 gene.
3. The combined detection method for avian immunosuppressive diseases based on multiplex fluorescence quantitative PCR according to claim 1, wherein in S12, the following conditions are satisfied when designing specific primers and fluorescent probes: the first condition is that no complementary pairing sequence exists between each primer pair and the probe, so that primer dimer or probe-primer complex is avoided; secondly, controlling the Tm value of each primer to be 58-62 ℃ and the Tm value of each probe to be 8-10 ℃ higher than that of the corresponding primer; Thirdly, marking different fluorescent reporter groups at the 5 'end of the probe, and marking a quenching group BHQ-1 at the 3' end; And in the fourth condition, the primer length is 18-25bp, and the probe length is 20-30bp.
4. The method for combined detection of avian immunosuppressive diseases based on multiplex fluorescence quantitative PCR according to claim 1, wherein the concentration of the positive standard in S23 is 。
5. The combined detection method for avian immunosuppressive diseases based on multiplex fluorescence quantitative PCR according to claim 1, wherein the specific steps of S41 are as follows: s411, taking 50 mu L of extracted nucleic acid sample, adding 10 mu L of nucleic acid purification buffer solution, and standing for 5-7min at room temperature after vortex oscillation for 10-12S; s412, adding 60 mu L of chloroform-isoamyl alcohol mixed solution, shaking vigorously for 30-32S, and centrifuging 12000r/min for 10-12min; s413, sucking the upper water phase into a new sterile centrifuge tube, adding equal volume of isopropanol, and standing at-20 ℃ for 20min to precipitate nucleic acid; S414, centrifuging at 12000r/min for 15-17min, discarding supernatant, washing the precipitate with 75% absolute ethanol for 2 times, and centrifuging at 12000r/min for 5min each time; s415, discarding ethanol, airing the precipitate at room temperature, and adding 20 mu L of enzyme-free pure water to dissolve the nucleic acid.
6. The method for combined detection of avian immunosuppressive diseases based on multiplex fluorescence quantitative PCR according to claim 5, wherein the volume ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol mixed solution in S412 is 24:1.
7. The combined detection method for avian immunosuppressive diseases based on multiplex fluorescence quantitative PCR according to claim 1, wherein in S42, the specific steps of the inhibitor residue detection are as follows: s421, adding an internal reference primer and a probe in the kit by taking the purified nucleic acid as a template, and carrying out single fluorescent quantitative PCR amplification; s422, if the Ct value of the reference gene is less than or equal to 32, the sample has no obvious inhibitor residue, and if the Ct value is more than 32 or no amplification curve, the purification treatment is needed again.
8. The combined detection method for avian immunosuppressive diseases based on multiplex fluorescence quantitative PCR according to claim 1, wherein in S5, the total volume of the reaction system is And each component and concentration includes: each pathogenic upstream primer is Each pathogenic downstream primer is Each pathogenic fluorescent probe is The template nucleic acid is The pure water without enzyme is complemented to 。
9. The method for combined detection of avian immunosuppressive diseases based on multiplex fluorescent quantitative PCR according to claim 1, wherein in S61, the reaction procedure is 95℃pre-denaturation for 5min, 95℃denaturation for 15S,60℃annealing for 30S, total of 40 cycles, 95℃melting curve analysis is performed for 15S,60℃for 1min, 95℃for 15S.
10. The method for combined detection of avian immunosuppressive diseases based on multiplex fluorescence quantitative PCR according to claim 1, wherein in the step S71, the analysis results are judged as negative control, blank control has no amplification curve, the detection result is valid when the positive control has a specific amplification curve and the melting curve has no impurity peak, the Ct value of the sample to be detected is less than or equal to 35, the melting curve is a single peak, the sample to be detected is judged as positive, the Ct value of the sample to be detected is >35 and the sample to be detected is rechecked when the sample to be detected is <40, the Ct value after rechecked is still >35, and the amplification curve has no amplification curve.

Description

Combined detection method for avian immunosuppressive diseases based on multiplex fluorescence quantitative PCR Technical Field The invention relates to the technical field of poultry disease detection, in particular to a poultry immune suppression disease combined detection method based on multiplex fluorescence quantitative PCR. Background Avian immunosuppressive diseases are a group of diseases which are mainly characterized by impairing the function of the poultry immune system and causing the body immunity to decline, and common diseases include Infectious Bursal Disease (IBD), marek's Disease (MD), reticuloendotheliosis (RE) of chickens, avian Leukemia (AL), infectious anemia (CIA) of chickens, and the like. The disease can directly cause the morbidity and mortality of poultry, and more seriously, the immune response capability of the poultry is reduced, so that the immune effect of the poultry on other vaccines is greatly reduced, and the poultry is secondarily infected by various bacteria and viruses, so that huge economic losses are brought to poultry farming industries such as chicken farming industry, duck farming industry and the like. Currently, the detection methods aiming at the avian immunosuppressive diseases are various, and the traditional detection methods comprise pathogen separation and identification and serological detection (such as ELISA (enzyme-linked immunosorbent assay), agar diffusion test and the like). Although the serological detection method is relatively simple in operation, the problems of poor specificity and insufficient sensitivity exist, and various diseases are difficult to detect simultaneously, so that missed diagnosis is easy to occur for sick poultry at early infection stage and the antibody level is not yet increased. Along with the development of molecular biology technology, the PCR technology is gradually applied to poultry disease detection, wherein the fluorescent quantitative PCR technology is widely applied to detection of various pathogens due to the advantages of strong specificity, high sensitivity, accurate quantification, high detection speed and the like. However, the existing detection methods of the avian immunosuppressive diseases based on fluorescence quantitative PCR are mostly single pathogen detection methods, namely, one method can only detect one kind of immunosuppressive disease pathogen. In an actual cultivation scene, the poultry often has mixed infection of multiple immunosuppressive pathogens, if a single pathogen detection method is adopted, multiple independent detections are required to be carried out on the same sample, so that the detection cost is increased, the detection time is prolonged, the waste of the sample is caused, the rapid screening of a large-scale sample is not facilitated, and the requirements of the cultivation industry on early stage, rapid and joint diagnosis of the avian immunosuppressive diseases are difficult to be met. In addition, the existing partial multiplex fluorescence quantitative PCR detection method does not fully consider the problem of mutual interference among different pathogenic primers in the design process, so that the conditions of nonspecific amplification, low amplification efficiency and the like occur in the detection process, the accuracy and the reliability of detection results are affected, and meanwhile, most detection methods do not establish a unified positive reference standard and quantitative analysis system, the detection results among different laboratories are difficult to compare, and the standardized application of detection data is not facilitated. Disclosure of Invention In order to solve the technical problems, the invention provides the following technical scheme: the combined detection method for the avian immunosuppressive diseases based on multiplex fluorescence quantitative PCR comprises the following specific steps: s1, determining a detection target and designing specific primers and probes: S11, according to the genome sequence of 5 common avian immunosuppressive disease pathogens of chicken Infectious Bursal Disease Virus (IBDV), chicken Marek' S disease virus (MDV), chicken reticuloendotheliosis virus (REV), avian Leukemia Virus (ALV) and Chicken Infectious Anemia Virus (CIAV), using bioinformatics software (such as NCBIBLAST and PrimerPremier 5.0) to conduct sequence alignment analysis, screening out highly conserved and highly specific gene fragments in each pathogenic genome, wherein IBDV takes VP2 gene as a target, MDV takes meq gene as a target, REV takes env gene as a target, ALV takes gp85 gene as a target, and CIAV takes VP1 gene as a target; S12, designing specific primers and fluorescent probes based on the sequences of the screened pathogenic target genes, wherein the design process needs to meet the following conditions that 1, no complementary pairing sequence exists between each primer pair and each probe to avoid forming primer dimers or probe-pr