Multiplex PCR (polymerase chain reaction) primer group for simultaneously detecting Newcastle disease virus, avian influenza virus, avian coronavirus and Rosella rosea and application of multiplex PCR primer group

Abstract

The invention belongs to the field of biological information detection, and provides a multiplex PCR (polymerase chain reaction) detection primer group for simultaneously detecting Newcastle disease virus, avian influenza virus, avian coronavirus and Rosella rosea and an application of the multiplex PCR detection primer group for simultaneously detecting Newcastle disease virus, avian influenza virus, avian coronavirus and Rosella rosea. The formation of a primer dimer or non-specific binding with other amplified fragments is prevented; the GC contents of the upstream primer and the downstream primer are similar, and more than three continuous G or C do not appear at the 3'end. The annealing temperatures of the primers are similar, the length difference of the target genes of the amplified fragments is distinguished through electrophoresis, and the length difference of the corresponding target genes among the primer groups is greater than or equal to 170bp; the kit can be used for quadruple PCR detection of the Newcastle disease virus, the avian influenza virus, the avian coronavirus and the Rosella murine, the simultaneous amplification and detection of the four pathogens are successfully realized, the result is accurate and stable, the identification degree is high, and the repeatability is good.

Description

Technical Field

[0001] This invention relates to the field of bioinformatics detection, and in particular to a multiplex PCR detection primer set for the simultaneous detection of four avian pathogens and its application, wherein the four avian pathogens are Newcastle disease virus, avian influenza virus, avian coronavirus and Rochetomyces ratiniformis. Background Technology

[0002] Newcastle disease is an acute, highly contagious, and deadly infectious disease caused by the Newcastle disease virus (NDV). It can infect various birds, including chickens, pigeons, wild birds, and other birds, causing enormous economic losses to the global poultry industry. The World Organisation for Animal Health (WOAH) classifies it as a Category A disease, while my country classifies it as a Category I animal disease.

[0003] Avian influenza is a highly contagious disease caused by the avian influenza virus (AIV), primarily affecting various poultry species such as chickens, ducks, and geese. Highly pathogenic avian influenza is a highly virulent infectious disease with a morbidity and mortality rate reaching 100%, while low pathogenic avian influenza can cause reduced growth and production performance and has been a disease with a relatively high incidence rate in recent years.

[0004] Avian coronaviruses (ACoVs) currently include the gamma and delta genera. Gamma coronaviruses primarily infect chickens, also known as infectious bronchitis virus (IBV), with typical symptoms including cough, difficulty breathing, swollen and pale kidneys with significant urate deposition. This is the most prevalent disease in chicks. Delta coronaviruses are mainly reported in birds and pigeons. my country has multiple migratory bird routes, and the habitats of birds, pigeons, waterfowl, and chickens overlap; therefore, long-term epidemiological surveillance of coronaviruses from these two genera is necessary.

[0005] Rothia nasimurium (RN) belongs to the Micrococcidae family of the genus Actinobacteria and is a facultative anaerobic, Gram-positive, opportunistic pathogenic coccus. Research on this bacterium is limited. Researchers have successively isolated Rothia nasimurium from clinical samples and diseased tissues of dogs, rabbits, sheep, geese, ducks, and chickens. Since 2024, the inventors have isolated four strains of this bacterium from the trachea of ​​chickens in two poultry farms with high mortality rates. Drug susceptibility testing showed that this bacterium exhibits zero inhibition zones against several commonly used antibiotics, including enrofloxacin, ciprofloxacin, cefotaxime, and kanamycin, demonstrating high drug resistance. Research on this bacterium is still scarce, lacking statistical data on its epidemiological surveys and clinical testing.

[0006] Currently, the detection of these four pathogens mainly relies on single-pathogen (RT-PCR) detection or quantitative real-time PCR. Using RT-PCR alone requires testing for each pathogen separately to determine whether a single or mixed infection exists in a sample. This not only results in long processing times, large workloads, and significant manpower and resource costs, but also increases detection costs. Quantitative real-time PCR reagents are more than 20 times more expensive than PCR, and the expensive equipment is not conducive to epidemiological surveys and clinical testing in poultry and birds. Therefore, providing a new method for the simultaneous detection of four pathogens—Newcastle disease virus, avian influenza virus, avian coronavirus, and *Rhodotorula musculosus*—is crucial for addressing the existing problems in pathogen surveillance.

[0007] Multiplex PCR is a pathogen detection method built upon single PCR. It involves adding multiple primer pairs to the same reaction system, requiring only one PCR amplification to detect multiple pathogens. Compared to single PCR, it offers advantages such as lower cost and faster detection. However, in practical applications, multiplex PCR presents technical challenges and requires meeting several requirements simultaneously. For example, interference may occur between different primer sets; non-specific binding may occur; inconsistent annealing temperatures between primer sets may result in some primer sets showing no bands or weak bands for the target gene; and if the target gene lengths amplified by different primer sets are similar, it may be difficult to distinguish the target genes after electrophoresis.

[0008] Therefore, developing a set of multiplex PCR detection primers that do not exhibit significant mutual interference, do not result in obvious nonspecific binding, have similar annealing temperatures, and exhibit large differences in target gene length is of great significance for achieving rapid and accurate multiplex PCR detection of various pathogens. Summary of the Invention

[0009] The purpose of this invention is to provide a multiplex PCR detection primer set for the simultaneous detection of Newcastle disease virus, avian influenza virus, avian coronavirus, and *Rhodotorula rubrum*, and its application, thereby filling a technological gap in the prior art. In the primer set design, continuous 3' end complementarity between primers is avoided to prevent primer dimer formation or non-specific binding with other amplified fragments. The GC content of the upstream and downstream primers should be similar (50%), and the 3' end should not contain more than three consecutive G or C molecules. The annealing temperatures of all primers should be similar (difference not exceeding 3°C). The difference in target gene length between amplified fragments should be distinguishable by electrophoresis, and the target gene length difference between primer sets should be ≥170 bp.

[0010] Using the provided multiplex PCR detection primer set, four pathogens can be accurately identified simultaneously in a single multiplex PCR reaction. Compared with the current traditional PCR technology, it is more accurate and efficient, and has high specificity and sensitivity.

[0011] This invention establishes a quadruple PCR detection primer set and corresponding detection method, improving PCR detection efficiency. The main challenge in achieving quadruple PCR detection lies in the design of the four primer sets. It is necessary to avoid mutual interference between primer sets, avoid non-specific binding of primers to the genomes of the four pathogens, and avoid non-specific binding of primers to the genomes of other avian pathogens. Furthermore, it is necessary to ensure that the annealing temperatures of each primer set are basically consistent, and that the target bands amplified by each set have sufficient length differences so that they can be clearly distinguished under different electrophoresis instruments and experimental environments. Moreover, the amplified target fragments, after DNA recovery and purification, can be used for sequence similarity analysis to determine the genotype (subtype) of the pathogen.

[0012] Under the above guiding principles, this invention first provides a multiplex PCR primer set for the simultaneous detection of Newcastle disease virus, avian influenza virus, avian coronavirus, and *Rhodotorula rubrum*, including upstream and downstream primers for Newcastle disease virus, avian influenza virus, avian coronavirus, and *Rhodotorula rubrum*, wherein: The upstream and downstream primers for Newcastle disease virus are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; The upstream and downstream primers for the avian influenza virus are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The upstream and downstream primers for avian coronaviruses are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. The upstream and downstream primers for *Rhodotorula musculosus* are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0013] All four primer pairs mentioned above were designed and screened by the inventors. The specific design and screening approach is as follows: *Rhodotorula musculosus* has a very high 16S rRNA gene similarity (above 98%), and this DNA gene is not a single gene within the bacteria but rather two 16S rRNA genes. Therefore, its detection is relatively easier compared to the other three gene-unstable RNA viruses. The inventors placed the primer design and screening for *Rhodotorula musculosus* last. The primer design and screening for the three gene-unstable RNA viruses—Newcastle disease virus, avian influenza virus, and avian coronavirus—was prioritized over the aforementioned DNA bacterial genomes. Before primer design, the inventors downloaded the complete genome sequences of the three pathogens from NCBI's GenBank: 90 strains of different genotypes of NDV, 200 strains of different subtypes of AIV, and 51 strains of ACoV. The nucleotide variation rate of the genes was analyzed, and genes with low variation frequencies were screened. The results showed that the nucleotide variation rate of the ACoV RdRp gene was 3.71 × 10⁻⁶. -4The substitutions / site / year and the AIV NP gene nucleotide variation rate were 1.37 × 10⁻⁶. -4 The substitutions / site / year and NDV NP gene nucleotide variation rate were 0.95 × 10⁻⁶. -4 Substitutions / site / year. The inventors further compared gene sequences to find 10 pairs of primer sequences each with high similarity, few variant nucleotides, and suitable primer annealing temperatures. These primers were then used in singleton and multiplex PCR with the recombinant plasmid to remove primer combinations with characteristics such as non-specific amplification, low sensitivity, and small differences in the length of the target gene. Finally, the primer sets with the best matching effect were selected.

[0014] Based on the aforementioned multiplex PCR detection primer set, the inventors also provided its application in detecting Newcastle disease virus, avian influenza virus, avian coronavirus, and Rochetomyces ratiniformis.

[0015] The specific steps are as follows: using the DNA (cDNA) of the sample to be tested as a template, a multiplex PCR amplification reaction is performed using the multiplex PCR primer set for simultaneous detection of Newcastle disease virus, avian influenza virus, avian coronavirus and rodenticide rotavirus. The products of the multiplex PCR amplification reaction are subjected to agarose gel electrophoresis. The presence or absence of the target band is used to determine whether the sample to be tested contains Newcastle disease virus, avian influenza virus, avian coronavirus and rodenticide rotavirus.

[0016] In some embodiments of the present invention, the system of the multiplex PCR amplification reaction is as follows: 12.5 μL of Taq PCR Mix premix, 0.5 μL each of the upstream and downstream primers for Newcastle disease virus, 0.5 μL each of the upstream and downstream primers for avian influenza virus, 0.6 μL each of the upstream and downstream primers for avian coronavirus, 0.5 μL each of the upstream and downstream primers for Rochetomyces roxburghii, 1-4 μL of DNA or cDNA nucleic acid template of the sample to be tested, and ddH2O to a final volume of 25 μL; The working concentration of each upstream and downstream primer is 10 μM.

[0017] In some embodiments of the present invention, the reaction program for the multiplex PCR amplification reaction is as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 50 s, for a total of 35 cycles; and extension at 72°C for 5 min. The product of the multiplex PCR amplification reaction is stored at 4°C.

[0018] The electrophoresis conditions required for the reaction products are as follows: 1.5%-2% agarose gel, DNA marker DL1000 or DL2000, Tris-boric acid (TBE) electrophoresis buffer, voltage 100V, and electrophoresis time 20-25min.

[0019] The results are judged as follows: if a target band of 582 bp is present, it indicates that the sample contains Newcastle disease virus; if a target band of 392 bp is present, it indicates that the sample contains avian influenza virus; if a target band of 204 bp is present, it indicates that the sample contains avian coronavirus; if a target band of 778 bp is present, it indicates that the sample contains Rochetomyces roxburghii. The presence of a single or mixed infection of the above four pathogens is determined based on the target band.

[0020] The inventors further claim protection for a multiplex PCR kit for the simultaneous detection of Newcastle disease virus, avian influenza virus, avian coronavirus, and *Rhodotorula rubrum*, the kit containing upstream and downstream primer sets for the aforementioned Newcastle disease virus, avian influenza virus, avian coronavirus, and *Rhodotorula rubrum*, in addition to a hot-start anti-interference Taq DNA polymerase and four recombinant plasmids as positive controls. The recombinant plasmids are pMD-NDV-NP, pMD-AIV-NP, pMD-ACoV-RdRp, and pMD-RN-16S positive plasmids. The pMD-NDV-NP, pMD-AIV-NP, pMD-ACoV-RdRp, and pMD-RN-16S positive plasmids are respectively recombinant plasmids of pMD18-T vector plasmid and NDV-NP582, AIV-NP392, AIV-RdRp204, and RN-16S778. NDV-NP582 is shown in SEQ ID NO. 9, and AIV-NP392 is shown in SEQ ID NO. 9. As shown in NO.10, ACov-RdRp204 is shown in SEQ ID NO.11, and RN-16S778 is shown in SEQ ID NO.12. All other reagent kit components are conventional choices in the art and will not be described further by the inventors.

[0021] The following beneficial effects can be achieved by using this invention to detect Newcastle disease virus, avian influenza virus, avian coronavirus, and Rochetomyces rodentata: This invention employs a multiplex PCR detection method and designs primer sets for Newcastle disease virus, avian influenza virus, avian coronavirus, and rodenticide *Rhodotorula rubrum*. By adding multiple primer sets to the same reaction, multiple pathogens can be detected simultaneously with a single PCR amplification. This method has the advantages of low cost and rapid detection. Compared with current traditional PCR techniques, it is more accurate and efficient, and has high specificity and sensitivity.

[0022] The multiplex PCR primer set for simultaneous detection of four avian pathogens provided by this invention exhibits no mutual interference between primer sets, no significant non-specific binding between primers or between primers and amplified fragments, and the annealing temperatures of each primer set are essentially consistent. Furthermore, the lengths of the target bands in each primer set vary sufficiently. Therefore, the primer set in this scheme can be used for quadruple PCR detection of Newcastle disease virus, avian influenza virus, avian coronavirus, and Rochetomyces rodentata, successfully achieving simultaneous amplification and detection of the aforementioned four pathogens. The results are accurate, stable, highly distinguishable, and have good reproducibility. Attached Figure Description

[0023] Figure 1 This is the result of a singleton PCR sensitivity test for Newcastle disease virus, where M is Marker DNA 2000, and 1-8 are 10. -1 -10 -8 The Newcastle disease virus plasmid template was diluted at 8 different dilutions, while the control group used ddH2O instead of the Newcastle disease virus plasmid template. Figure 2 These are the results of singleton PCR sensitivity testing for avian influenza virus, where M represents Marker DNA2000, and 1-8 represent 10. -1 -10 -8 The avian influenza virus plasmid template was diluted at 8 different dilutions, while the control group used ddH2O instead of the avian influenza virus plasmid template. Figure 3 These are the results of singleton PCR sensitivity testing for avian coronaviruses, where M represents Marker DNA2000, and 1-8 represent 10... -1 -10 -8 The avian coronavirus plasmid template was diluted at 8 different dilutions, with the control group consisting of ddH2O instead of the avian coronavirus plasmid template. Figure 4 These are the results of singleton PCR sensitivity testing for Rochetomyces ratina, where M represents Marker DNA2000, and 1-8 represent 10. -1 -10 -8 Eight dilutions of the *Rhodotorula musculosus* plasmid template were used, with the control group consisting of *Rhodotorula musculosus* plasmid template replaced by ddH2O. Figure 5 These are the results of multiplex PCR specific detection, where M is Marker DNA2000, 1 is ACoV; 2 is AIV; 3 is NDV; 4 is RN; 5 is a mixed template of four standard plasmids in equal volumes; 6 is ILTV; 7 is FPV; 8 is aMPV; 9 is MG; 10 is MS; 11 is APEC-O2 strain; and 12 is Hpg. Figure 6The results are from multiplex PCR sensitivity testing, where M is Marker DNA2000 and 1-8 are templates of four standard plasmids mixed in equal volumes at 10-fold serial dilutions. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to specific embodiments. These embodiments are only used to illustrate the technical solution of the present invention in more detail and should not be construed as limiting the scope of protection of the present invention.

[0025] Unless otherwise specified, all substances and reagents used in the following examples are generally commercially available products; the reagents or kits used are used in accordance with conventional methods in the art or as recommended in the instructions.

[0026] The preferred multiplex PCR reaction system includes: 12.5 μL of Taq PCR Mix premix, 0.5 μL each of the upstream and downstream primers for Newcastle disease virus, 0.5 μL each of the upstream and downstream primers for avian influenza virus, 0.6 μL each of the upstream and downstream primers for avian coronavirus, 0.5 μL each of the upstream and downstream primers for Rochetomyces ratina, 1-4 μL of DNA (cDNA) nucleic acid template or plasmid template of the sample to be tested, and ddH2O to a final volume of 25 μL.

[0027] The preferred multiplex PCR reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 35 cycles; 72℃ extension for 5 min.

[0028] The products of the multiplex PCR amplification reaction were stored at 4°C.

[0029] The required electrophoresis conditions for the reaction products are as follows: 1.5%-2% agarose gel, DNA marker DL1000 or DL2000, Tris-boric acid (TBE) electrophoresis buffer, voltage 100V, and electrophoresis time 20-25 min. After electrophoresis, the presence of a target band of 582 bp indicates the presence of Newcastle disease virus in the sample; a target band of 392 bp indicates the presence of avian influenza virus; a target band of 204 bp indicates the presence of avian coronavirus; and a target band of 778 bp indicates the presence of *Rhodotorula rubrum*. The presence of a single or mixed infection of these four pathogens is determined based on the target bands.

[0030] Example 1 Primer Design and Synthesis 1. Based on the NP gene sequences of different genotypes of Newcastle disease (NDV) in Class II published in GenBank, nucleotide regions with high similarity were selected. PCR primers for detecting NDV were designed using Primer 5.0 software and synthesized by BGI Genomics Co., Ltd. The primers can amplify a length of 582 bp. The primer set sequence is as follows: NDV-582F, a specific upstream primer for NDV: 5'-CGATGATAGCAGGATCTCTC-3'(SEQ ID NO.1); NDV-582R, a specific downstream primer for NDV: 5'-CAAAGCTCATCTGGTCACTA-3' (SEQ ID NO. 2).

[0031] 2. Based on the NP gene sequences of different subtypes of avian influenza virus (AIV) published in GenBank, nucleotide regions with high similarity were selected. PCR primers for detecting AIV were designed using Primer 5.0 software. These primers, synthesized by BGI Genomics Co., Ltd., can amplify a length of 392 bp. The primer set sequence is as follows: AIV-specific upstream primer AIV-392F: 5'-TTTAGGRTTTGTGTTCACGC-3' (SEQ ID NO. 3); AIV-specific downstream primer AIV-392R: 5'-CCATAGCCTTAGCCGTAGTA-3' (SEQ ID NO. 4).

[0032] 3. Based on the RdRp gene sequences of avian coronaviruses ACoV (γ and δ genera) published in GenBank, highly similar nucleotide regions were selected. PCR primers for detecting ACoV were designed using Primer 5.0 software and synthesized by BGI Genomics Co., Ltd. These primers amplify a length of 204 bp. The primer set sequence is as follows: ACoV-specific upstream primer ACoV-204F: 5'-ACTATCCTAAATGTGATAGAGCC-3' (SEQ ID NO.5); ACoV-specific downstream primer ACoV-204R: 5'-TGGCATCACCACTGCTAGTACCA-3' (SEQ ID NO. 6).

[0033] 4. Based on the 16S rRNA gene sequences of duck, goose, and human *Rhizobium nervosa* (RN) published in GenBank, and the 16S rRNA gene sequences of four chicken-derived *Rhizobium nervosa* strains sequenced in our laboratory, PCR primers for detecting *Rhizobium nervosa* were designed using Primer 5.0 software. These primers, synthesized by BGI Genomics Co., Ltd., can amplify a length of 778 bp. The primer set sequence is as follows: RN-specific upstream primer Rothia-778F: 5'- CCTGGTGTAGCGGTGAAAT -3' (SEQ ID NO. 7); RN-specific downstream primer Rothia-778R: 5'-ACAAGGGGTTAGGCCATCG-3' (SEQ ID NO. 8).

[0034] Example 2 Construction of plasmid standards DNA from Newcastle disease virus (NDV), avian influenza virus (AIV), and avian coronavirus (ACoV) was extracted according to the instructions of the viral DNA / RNA extraction kit (Simply P Virus DNA / RNA Co-extraction Kit, Hangzhou Bori Technology Co., Ltd.), and then used a reverse transcription kit (PrimeScript) to extract DNA. TM IV 1st strand cDNA Synthesis Mix (Baori Biotechnology (Beijing) Co., Ltd.) reverse transcribes RNA into cDNA; DNA was extracted from *Rhodotorula musculosus* (RN) using a bacterial genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.).

[0035] Using primers SEQ ID NO.1-8, the target gene was amplified by PCR using cDNA (DNA) of four pathogens as templates and corresponding primers, and the target fragment was recovered. The pMD18-T vector cloning kit (purchased from Baori Biotechnology (Beijing) Co., Ltd.) was used to ligate the target fragment overnight at 4°C, and then transformed into DH5α competent cells according to the instructions. The cells were then spread onto LB agar plates containing ampicillin (final concentration 50 μg / ml - 100 μg / ml) and cultured for 16-20 hours. Single colonies were picked for PCR identification. Positive single colonies were inoculated into liquid LB medium containing ampicillin (final concentration 100 μg / ml) for overnight expansion. Recombinant plasmids were extracted using a plasmid extraction kit purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0036] The sequences were subjected to BLAST alignment analysis. The recombinant plasmids pMD-NDV-NP, pMD-AIV-NP, pMD-ACoV-RdRp, and pMD-RN-16S positive plasmids that were correctly identified by sequencing were used as standards. The concentration and copy number were determined using an ultraviolet spectrophotometer and stored at -20℃ for later use. Among them, NDV-NP582 is shown in SEQ ID NO.9, AIV-NP392 is shown in SEQ ID NO.10, ACov-RdRp204 is shown in SEQ ID NO.11, and RN-16S778 is shown in SEQ ID NO.12.

[0037] The standard positive plasmid is calculated based on the copy number formula: (6.02 × 10⁻⁶) 23 )×(plasmid concentration×10 -9 ) / (DNA length × 660) = copies / μL, plasmid concentration is in ng / μL. The calculated copy number is: NDV is 3.7 × 10⁻⁶. 8 copies / μL, AIV 1.69×10 8 copies / μL, ACoV 6.17×10 8 copies / μL, RN 2.33×10 8 copies / μL.

[0038] Example 3 Nucleic Acid Extraction and Reverse Transcription RNA / DNA of the following viruses (all commercially available standard strains) were extracted according to the instructions of the Viral DNA / RNA Extraction Kit (Simply P Virus DNA / RNA Co-extraction Kit, Hangzhou Bori Technology Co., Ltd.) (Newcastle disease virus (NDV), avian influenza virus (AIV), avian coronavirus (ACoV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), and avian metapneumovirus (aMPV)). The RNA / DNA of these viruses was then extracted using a reverse transcription kit (PrimeScript). TM IV 1st strand cDNA SynthesisMix (Baori Biotechnology (Beijing) Co., Ltd.) reverse transcribes RNA into cDNA; DNA was extracted from bacteria (Rhodotorula musculosus (RN), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), avian pathogenic Escherichia coli (APEC-02), and Haemophilus paragallinarum (Hpg)) using a bacterial genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). All of the above strains are common strains in this field and can be purchased directly from the market or obtained from cultural relics centers; the inventors will not elaborate further.

[0039] The extracted viral RNA reverse transcription product, viral DNA, and bacterial DNA were stored at -80℃ for later use.

[0040] Example 4: Multiplex PCR kit for simultaneous detection of Newcastle disease virus, avian influenza virus, avian coronavirus, and Rochetomyces rodentata. This kit contains upstream and downstream primer sets for Newcastle disease virus, avian influenza virus, avian coronavirus, and *Rhodotorula rubrum*, as described above. In addition, it contains hot-start anti-interference Taq DNA polymerase and four recombinant plasmids as positive controls. The recombinant plasmids are pMD-NDV-NP, pMD-AIV-NP, pMD-ACoV-RdRp, and pMD-RN-16S positive plasmids as described in Example 2. All other kit components are conventionally selected in the art.

[0041] Experimental Example 1: Optimization of Multiplex PCR Reaction Conditions Using the recombinant plasmid from Example 2 diluted 1000 times as a template, the primer concentration in the single-component system was explored according to the PCR reaction conditions.

[0042] First, the annealing temperature was screened. PCR reactions were carried out according to the primer concentration, reaction system and reaction conditions recommended in the Taq PCR premix instructions (Beijing TransGen Biotech Co., Ltd., 2×Easy Taq PCR Super Mix). The annealing temperatures were 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃ and 58℃, respectively.

[0043] Then, primer concentration screening was performed, and primers with a working concentration of 10 μM were prepared for each group. In the aforementioned 25 μL system, the upstream and downstream primers were added in equal volumes, with each primer added in a series of volumes of 0.3 μL, 0.4 μL, 0.5 μL, 0.6 μL, 0.7 μL, and 0.8 μL, respectively. The optimal PCR reaction conditions were then screened and optimized to obtain the best reaction conditions.

[0044] Based on the individual optimization of the quadruple system, the mixed primers and annealing extension temperature were further optimized to obtain the optimal multiplex PCR reaction system and reaction conditions as follows: 0.5 μL each of the upstream and downstream primers for Newcastle disease virus, 0.5 μL each of the upstream and downstream primers for avian influenza virus, 0.6 μL each of the upstream and downstream primers for avian coronavirus, 0.5 μL each of the upstream and downstream primers for Rochetomyces roxburghii, and an annealing temperature of 55 °C.

[0045] Experiment Example 2: Specificity Experiment Using DNA or cDNA of Newcastle disease virus (NDV), avian influenza virus (AIV), avian coronavirus (ACoV), Rasulella vesicatoria (RN), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), avian metapneumovirus (aMPV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), avian Escherichia coli (APEC-O2 strain), and Haemophilus paragallinarum (Hpg) as templates, PCR amplification was performed under optimized conditions. The optimized conditions were: The multiplex PCR reaction system includes: 12.5 μL of Taq PCR Mix premix, 0.5 μL each of the upstream and downstream primers for Newcastle disease virus, 0.5 μL each of the upstream and downstream primers for avian influenza virus, 0.6 μL each of the upstream and downstream primers for avian coronavirus, 0.5 μL each of the upstream and downstream primers for Rochetomyces ratina, 1-4 μL of DNA (cDNA) template or mixed plasmid template of the sample to be tested, and ddH2O to a final volume of 25 μL.

[0046] Table 1 Reaction System The reaction program for multiplex PCR was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 35 cycles; 72℃ extension for 5 min.

[0047] The required electrophoresis conditions for the reaction products are as follows: 1.5%-2% agarose gel, DNA marker DL1000 or DL2000, Tris-boric acid (TBE) electrophoresis buffer, voltage 100V, and electrophoresis time 20-25 min. After electrophoresis, the presence of a target band of 582 bp indicates the presence of Newcastle disease virus in the sample; a target band of 392 bp indicates the presence of avian influenza virus; a target band of 204 bp indicates the presence of avian coronavirus; and a target band of 778 bp indicates the presence of *Rhodotorula rubrum*. The presence of a single or mixed infection of these four pathogens is determined based on the target bands.

[0048] The specificity of this method is determined by whether the target fragment is present after gel electrophoresis. Results are shown below. Figure 5 As can be seen from the figure, the PCR identification method based on primer set construction provided by this invention has good specificity.

[0049] Experiment Example 3: Single-Sensitivity Experiment The sensitivity of PCR detection was determined by serially diluting the concentrations of four plasmid standards tenfold. The four recombinant plasmids pMD-NDV-NP, pMD-AIV-NP, pMD-ACoV-RdRp, and pMD-RN-16S from Example 2 were each serially diluted 10-fold. -1 -10 -8 Using eight dilutions as templates, single-dose sensitivity experiments were performed on pMD-NDV-NP, pMD-AIV-NP, pMD-ACoV-RdRp, and pMD-RN-16S respectively (i.e., the system contained only one of the aforementioned four recombinant plasmids and its corresponding primer) to detect sensitivity.

[0050] PCR amplification was performed under optimized reaction conditions (refer to Experimental Example 2). After amplification, the products were subjected to gel electrophoresis as described in Experimental Example 2. The minimum detectable copy number was determined based on the presence or absence of the target band in the reaction product. The results are as follows: Figure 1-4 The results showed that, based on the single-pass sensitivity experiment, the lowest detectable copy number was NDV of 3.7 × 10⁻⁶. 3 copies / μL, AIV is 1.69×10 3 The number of copies / μL for ACoV was 6.17 × 10⁻⁶. 3 copies / μL, RN is 2.33×10 2 copies / μL.

[0051] Experiment Example 4: Multiple Sensitivity Experiment The optimized reaction conditions were followed, and the multiplex PCR reaction system consisted of: 12.5 μL of Taq PCR Mix premix, 0.5 μL each of the upstream and downstream primers for Newcastle disease virus, 0.5 μL each of the upstream and downstream primers for avian influenza virus, 0.6 μL each of the upstream and downstream primers for avian coronavirus, 0.5 μL each of the upstream and downstream primers for Rochetomyces ratina, 1 μL of each of the four recombinant plasmid nucleic acid templates, and ddH2O to a final volume of 25 μL. Multiplex sensitivity experiments were performed to determine the sensitivity.

[0052] PCR amplification was performed under an optimized PCR program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 35 cycles; 72℃ extension for 5 min. After amplification, the products were subjected to gel electrophoresis according to the method in Experiment 2. A 1.5%-2% agarose gel was prepared, and the DNA marker used was DL1000 or DL2000, with Tris-boric acid (TBE) electrophoresis buffer, a voltage of 100V, and an electrophoresis time of 20-25 min. The minimum detectable copy number of this multiplex PCR method was determined based on the presence or absence of the target band in the reaction product. The results are as follows: Figure 6The results showed that the lowest detectable copy number was NDV 3.7 × 10⁻⁶. 3 copies / μL, AIV is 1.69×10 3 The number of copies / μL for ACoV was 6.17 × 10⁻⁶. 3 copies / μL, RN is 2.33×10 3 copies / μL.

[0053] Experimental Example 5: Clinical Sample Testing A total of 215 pharyngeal swabs from patients with respiratory diseases were collected from different poultry farms. The primer set and corresponding quadruple PCR detection method provided in this application were used to detect them. Simultaneously, single PCR detection was performed using the primers provided in this application for verification. The detection results of the three viral diseases in the above samples were compared by a laboratory that has passed the China National Accreditation Service for Conformity Assessment (CNAS) using the conventional PCR or qPCR method in the national standard, and the concordance rate was calculated.

[0054] Table 2. Concordance rates of quadruple PCR and single PCR on clinical samples. The test results showed that for Newcastle disease pathogen detection, the quadruple PCR method had a 100% concordance rate with both single PCR and CNAS laboratory results; for AIV detection, the positive rates were 22.79% (49 / 215) for the quadruple PCR method, 24.19% (52 / 215) for single PCR, and 24.65% (53 / 215) for CNAS laboratory, with the quadruple PCR method showing a concordance rate higher than 98.14% with the other two methods; for ACoV detection, the positive rates were 35.81% (77 / 215) for the quadruple PCR method, 37.67% (81 / 215) for single PCR, and 38.6% (83 / 215) for CNAS laboratory, with the quadruple PCR method showing a concordance rate higher than 97.21% with the other two methods.

[0055] The above results indicate that the quadruple PCR detection method has a very high concordance rate with single PCR or CNAS detection results, and has the advantage of detecting four pathogens in one reaction, making it suitable for epidemiological detection studies of four pathogens.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any equivalent substitutions or modifications made by those skilled in the art within the spirit and principle of the present invention without any creative effort should be included within the protection scope of the present invention.

Claims

1. A multiplex PCR primer set for simultaneous detection of Newcastle disease virus, avian influenza virus, avian coronavirus, and *Rhodotorula musculosus*, characterized in that: This includes upstream and downstream primers for Newcastle disease virus, avian influenza virus, avian coronavirus, and *Rhodotorula rubrum*, among which: The upstream and downstream primers for Newcastle disease virus are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; The upstream and downstream primers for the avian influenza virus are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The upstream and downstream primers for avian coronaviruses are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. The upstream and downstream primers for *Rhodotorula musculosus* are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

2. The multiplex PCR primer set according to claim 1, characterized in that, The corresponding multiplex PCR amplification reaction system is as follows: 12.5 μL Taq PCR Mix premix, 0.5 μL each of the upstream and downstream primers for Newcastle disease virus, 0.5 μL each of the upstream and downstream primers for avian influenza virus, 0.6 μL each of the upstream and downstream primers for avian coronavirus, 0.5 μL each of the upstream and downstream primers for Rochetomyces roxburghii, 1-4 μL of DNA or cDNA nucleic acid template from the sample to be tested, and ddH2O to a final volume of 25 μL. The working concentration of each upstream and downstream primer is 10 μM.

3. The multiplex PCR primer set according to claim 1 or 2, characterized in that, The corresponding multiplex PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 50 s, for a total of 35 cycles; 72℃ extension for 5 min; the multiplex PCR amplification reaction products are stored at 4℃.

4. The multiplex PCR primer set according to claim 3, characterized in that, The electrophoresis conditions for the reaction products are as follows: 1.5%-2% agarose gel, DNA marker DL1000 or DL2000, Tris-boronic acid electrophoresis buffer, voltage 100V, electrophoresis time 20-25min.

5. A multiplex PCR kit for simultaneous detection of Newcastle disease virus, avian influenza virus, avian coronavirus, and *Rhodotorula rubrum*, characterized in that, Containing the upstream and downstream primer sets of Newcastle disease virus, avian influenza virus, avian coronavirus, and Rochetomyces ratiniformis as described in claim 1, and further containing hot-start anti-interference Taq DNA polymerase and four recombinant plasmids as positive controls, wherein the recombinant plasmids are pMD-NDV-NP, pMD-AIV-NP, pMD-ACoV-RdRp, and pMD-RN-16S positive plasmids.

Images