Kit capable of simultaneously detecting eight porcine respiratory pathogens and application thereof

Abstract

The invention relates to the technical field of molecular biological detection, and particularly discloses a kit capable of simultaneously detecting eight porcine respiratory pathogens and application. The invention specifically relates to detection of hog cholera virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, circovirus type 2, African hog cholera virus, swine influenza A virus, streptococcus suis and swine pasteurella multocida. According to the combined detection composition provided by the invention, a dual quadruple fluorescent PCR analysis method is utilized, and different pathogens are detected by detecting target spots on the different pathogens, so that target genes of eight pathogens can be rapidly detected at a time, and detection and differential diagnosis are more efficient, economical, simple and convenient; the kit has the advantages of high sensitivity, good specificity, rapidness, high flux, simplicity and convenience in operation and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology detection, and particularly to a dual quadruplex fluorescence PCR primer-probe combination for simultaneously detecting 8 porcine respiratory pathogens, method establishment and application. Background Art

[0002] Classical Swine Fever Virus (CSFV) belongs to the genus Pestivirus of the family Flaviviridae, and is a single-stranded positive-strand RNA virus with an envelope structure. CSFV is the pathogen that causes Classical Swine Fever (CSF), which causes pigs to develop acute febrile systemic septicemic diseases, has high infectivity, a wide range of epidemics, and outbreaks occur all over the world, causing huge economic losses to the pig industry. The World Organization for Animal Health lists it as a disease that must be reported, and it is one of the most harmful and highly regarded diseases in pigs. Currently, there is no highly effective drug treatment method for classical swine fever, and prevention mainly relies on vaccination. Through vaccination with CSFV vaccines and pathogen purification strategies, many countries and regions have achieved the purification of this disease. However, CSFV still prevails in wild boar populations and outbreaks occur from time to time, putting domestic pigs at risk of infection.

[0003] Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the genus Arterivirus of the family Arteriviridae, and is a single-stranded positive-strand, non-segmented, enveloped RNA virus. It can cause infectious diseases characterized by reproductive disorders such as abortion, stillbirth, and mummified fetuses in sows and respiratory symptoms in pigs at all stages, and is also known as the "blue ear disease" virus. Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease that can damage the immune system and cause immunosuppression in the body. At the same time, it can induce mixed infections of various viruses, bacteria, and parasites, exacerbating the condition. Currently, many pig farms have latent infections of PRRSV. Due to the complex epidemic situation and severe mixed infections, it is very difficult for pig farms infected with PRRSV to be purified, and so far, no effective method to eradicate PRRS has been found.

[0004] Pseudorabies virus (PRV) is the pathogen of pseudorabies (PR). It can infect a variety of animals including pigs, sheep, ruminants, carnivores, and rodents, mainly causing nervous system diseases, respiratory system diseases, and reproductive failure. PRV is an enveloped linear double-stranded DNA virus and is a member of the Herpesviridae family, Alphaherpesvirinae subfamily, and Varicellovirus genus. After entering the pig body, PRV mainly causes neurological symptoms by invading the central nervous system. At the same time, it can also cause symptoms such as fever, difficulty breathing, and reproductive disorders. The virus enters the brain through the olfactory nerve, resulting in encephalitis and myelitis. The main source of infection of this disease is virus-carrying pigs in farms, especially pigs carrying the virus during the incubation period and pigs continuously carrying the virus after recovery. The virus can be transmitted vertically, resulting in piglets being naturally infected after birth. Direct contact between diseased pigs and healthy pigs, or contact with secretions and excretions discharged from diseased pigs, as well as sharing feed, drinking water, and breeding management tools can all achieve the rapid spread of the virus. Pigs of all ages are susceptible to porcine pseudorabies virus. Piglets, especially newborn piglets, are more susceptible to infection due to the incomplete development of their immune systems, and the lethality rate can reach 100%. In many farms, porcine pseudorabies virus does not have a specific epidemic season, and infections and transmissions can occur throughout the year. High stocking density, frequent contact, lack of an effective vaccination program, the presence of virus-carrying pigs, lack of strict biosafety measures within the farm, failure to effectively isolate diseased pigs, and failure to thoroughly disinfect pig houses will all promote or exacerbate the occurrence and epidemic of the disease.

[0005] Porcine circovirus (PCV) belongs to the genus Circovirus of the family Circoviridae. Four genotypes have been identified so far and are named PCV1-4 in consecutive numbers according to the order of their discovery. Currently, it is generally believed that PCV1 is non-pathogenic; since PCV3 was first discovered in 2016, it has been detected in many countries or regions and has a relatively high infection rate; PCV4 has only been reported in China and there is relatively little research on it at present. PCV2 is one of the most serious pathogens threatening the global pig industry. The infection of porcine circovirus type 2 mainly presents as postweaning multisystemic wasting syndrome, characterized by progressive emaciation, dyspnea, pale skin, enlarged lymph nodes and jaundice, and can also cause reproductive disorders, such as abortion, stillbirth, mummified fetus, weak fetus and other phenomena in sows. The mixed infection rate of porcine circovirus type 2 and pseudorabies virus in pig farms in some parts of China is as high as 60%, much higher than the single infection rate. Mixed infection will also lead to the aggravation of clinical symptoms and the mortality rate can reach 20%-30%, while the mortality rate of piglets caused by single infection of porcine circovirus type 2 is only 10%-20%. This is because the synergistic effect of the two viruses exacerbates the damage to the body and increases the risk of secondary infection, thus leading to a higher mortality rate. The clinical symptoms of porcine circovirus disease and pseudorabies are complex and diverse, often lacking specificity, and the symptoms are even more complex during mixed infection, posing a great challenge to clinical diagnosis. In addition, mixed infection will also increase the risk of secondary infection and the occurrence of other pathogen infections, further increasing the complexity of diagnosis.

[0006] African swine fever virus (ASFV) is a kind of enveloped, large, linear double-stranded DNA virus and is the only member of the family Asfarviridae. It is a highly contagious pathogen that causes African swine fever (ASF). Domestic pigs of all ages are susceptible and the mortality rate can reach 100%. Since ASFV was first reported in Kenya in 1921, it has caused widespread epidemics in many countries and regions in Africa, Europe, South America and Asia. Since it was introduced into China in 2018, ASF has gone through 4 main epidemic stages: highly virulent genotype II strains, gene-deleted strains, genotype I strains, and I / II recombinant strains. Currently, the prevalent strains of ASF in China show the characteristics of "multiple lineages, multiple time and space", posing new challenges to the prevention and control of ASFV. The clinical characteristics of African swine fever mainly include high fever, cyanosis of the skin, bleeding in all organs of the body, and the mortality rate is as high as 100%, posing a serious threat to the healthy pigs in farms. African swine fever is extremely harmful and has a variety of transmission routes. Once a pig is infected with African swine fever, a large amount of virus will be contained in its feces and secretions, posing a serious threat to the global pig industry. The prevention and control of African swine fever depends on clarifying its pathogenic mechanism and epidemiological variability. Due to the lack of safe and effective vaccines and antiviral drugs, culling is the most effective method to control the epidemic.

[0007] Swine Influenza Virus (SIV) belongs to the Orthomyxoviridae family and the Influenza virus genus. It is a pathogen that can cause swine influenza, and this virus is highly contagious. Once pigs are infected, it will cause significant economic losses to pig farms. After pigs are infected with the swine influenza virus, a series of symptoms will appear, mainly including coughing, fever, loss of appetite, rapid breathing, and listlessness. The swine influenza virus can affect the respiratory system of pigs, leading to symptoms such as dyspnea and rapid breathing in diseased pigs. After being infected with the swine influenza virus, the common symptom of diseased pigs is coughing, which usually lasts for several days. When the infection with the swine influenza virus is relatively severe, it may also cause serious complications in diseased pigs, such as pancytopenia, pleural effusion, severe pneumonia, renal failure, and even death. Like other type A influenza viruses, the genes of the swine influenza virus are unstable. In particular, nucleotide point mutations, substitutions, deletions, and insertions in the genomic sequences encoding HA and NA may lead to differences in amino acid translation at molecular antigenic sites and the emergence of new subtypes. Currently, various subtypes of SIV have been reported to be prevalent in pig populations worldwide, including subtypes such as H1N1, H1N2, H3N2, H5N1, H7N7, and H9N2, among which H1N1, H1N2, and H3N2 are the main prevalent subtypes. Pig respiratory epithelial cells simultaneously have SAα-2,3Gal and SAα-2,6Gal receptors, making them a common susceptible host for avian influenza virus, swine influenza virus, and human influenza virus, and becoming a natural "mixing vessel" for gene recombination, gene mutation, and cross-species transmission of different subtypes of influenza virus. Therefore, continuous monitoring of the prevalence of the swine influenza virus is of great significance.

[0008] Streptococcus suis (SS) is a Gram-positive bacterium that can cause diseases such as meningitis, septicemia, and arthritis in pigs. It is an important pathogenic bacterium in pigs, causing serious economic losses to the pig industry. At the same time, this bacterium can also infect humans, leading to meningitis, septicemia, and even death, making it an important zoonotic pathogen. Streptococcus suis disease can occur throughout the year without obvious seasonality. It is more likely to cause disease in hot seasons or places with poor sanitary conditions, especially with a relatively high incidence rate from May to November. Streptococcus suis can be transmitted through multiple routes. It can be vertically transmitted through sow production, lactation, etc., and can also be horizontally transmitted through skin wounds, mouth, nose, etc. Insect vectors are also one of the important transmission routes. Flies can mechanically carry streptococcus for about 3 days while maintaining its pathogenicity. Pigs of all ages can be infected with Streptococcus suis, but weaned piglets and fattening pigs are the most susceptible, especially piglets aged 3 to 12 weeks. Pregnant sows are also an important susceptible group. After infection, it can lead to abortion and the birth of dead fetuses. According to the differences in capsular polysaccharide antigens, Streptococcus suis can be divided into 29 traditional serotypes (1-19, 21, 23-25, 27-31, 1 / 2), Chz serotypes, and 33 new capsular gene clusters (NCL1-20, 21a, 21b, 22-32). In addition, there are also some untyped strains. Up to now, 11 serotypes of Streptococcus suis that have been reported to infect humans are serotype 1, 2, 4, 5, 7, 9, 14, 16, 21, 24, and 31. In 1968, Denmark first reported a case of Streptococcus suis infecting humans, and subsequently, there have been reports of Streptococcus suis infecting humans in regions such as Asia, Europe, South America, North America, Oceania, and Africa.

[0009] Pasteurella multocida (PM) is a highly contagious zoonotic Gram-negative bacterium belonging to the genus Pasteurella in the family Pasteurellaceae. PM can infect humans, various economic animals, and wild animals, seriously endangering the global livestock economy and threatening biosafety and human health. The extensive pathogenicity of PM is due to the complex pathological processes it induces in the respiratory system and other multiple systems, as well as bacteremia, etc. According to the capsular and lipopolysaccharide (LPS) antigens, PM can be divided into 5 capsular serotypes (A, B, D, E, F) and 16 LPS serotypes (1 - 16). Serotyping methods help determine the specific disease types caused by different strains after infecting hosts of different species. For example, capsular types A and D of PM usually cause swine respiratory diseases. Capsular types A and D of PM are the main serotypes that infect pigs and cause diseases, resulting in swine plague (non-toxigenic PM of type A) and atrophic rhinitis (toxigenic PM of type D). Clinically, the most common is the acute type, which causes inflammation, swelling of the pig's throat, difficulty breathing, weakness, and even suffocation and death. The chronic type is rare and the symptoms are not obvious, while the most acute type presents as septicemia. Studies on the pathogen composition and frequency of swine respiratory disease syndrome have shown that PM is the most common secondary pathogen, causing huge economic losses in infected animals from weight loss to death.

[0010] The above-mentioned classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus type 2, African swine fever virus, influenza A virus of swine, Streptococcus suis, and Pasteurella multocida can all cause swine respiratory syndrome, causing huge losses to the pig farming industry. Therefore, early diagnosis of pathogens and timely and effective anti-infection treatment and prevention and control can greatly reduce the morbidity and mortality. However, it is very difficult to identify and determine the types of infected pathogens through clinical symptoms and conventional laboratory tests, and multiple mixed infections pose difficulties for diagnosis.

[0011] Therefore, there is an urgent need in this field for a simple, rapid, and objective method for detecting the above-mentioned pathogens, so as to achieve clear identification of pathogens, early diagnosis, reasonable guidance for anti-infection treatment, and formulation of effective prevention and control measures. Summary of the Invention

[0012] In view of the above-mentioned existing technical defects, the present invention provides a kit and application for simultaneously detecting 8 swine respiratory pathogens.

[0013] A biochemical kit, the kit comprising the following primers and probes:

[0014] Primer ID NO: 1, ID NO: 2 and probe ID NO: 3;

[0015] Primer ID NO: 4, ID NO: 5 and probe ID NO: 6;

[0016] Primer ID NO: 7, ID NO: 8 and Probe ID NO: 9;

[0017] Primer ID NO: 10, ID NO: 11 and Probe ID NO: 12;

[0018] Primer ID NO: 13, ID NO: 14 and Probe ID NO: 15;

[0019] Primer ID NO: 16, ID NO: 17 and Probe ID NO: 18;

[0020] Primer ID NO: 19, ID NO: 20 and Probe ID NO: 21;

[0021] Primer ID NO: 22, ID NO: 23 and Probe ID NO: 24.

[0022] The present invention protects the application of the above kit in detecting porcine respiratory pathogens.

[0023] The porcine respiratory pathogens described in the present invention include classical swine fever virus and / or porcine reproductive and respiratory syndrome virus and / or pseudorabies virus and / or porcine circovirus type 2 and / or African swine fever virus and / or influenza A virus of swine and / or Streptococcus suis and / or Pasteurella multocida.

[0024] Detect classical swine fever virus using Primer ID NO: 1, ID NO: 2 and Probe ID NO: 3;

[0025] Detect porcine reproductive and respiratory syndrome virus using Primer ID NO: 4, ID NO: 5 and Probe ID NO: 6;

[0026] Detect pseudorabies virus using Primer ID NO: 7, ID NO: 8 and Probe ID NO: 9;

[0027] Detect porcine circovirus type 2 using Primer ID NO: 10, ID NO: 11 and Probe ID NO: 12;

[0028] Detect African swine fever virus using Primer ID NO: 13, ID NO: 14 and Probe ID NO: 15;

[0029] Detect influenza A virus of swine using Primer ID NO: 16, ID NO: 17 and Probe ID NO: 18;

[0030] Detect Streptococcus suis using Primer ID NO: 19, ID NO: 20 and Probe ID NO: 21;

[0031] Detect Pasteurella multocida in pigs using primer ID NO: 22, ID NO: 23 and probe ID NO: 24.

[0032] The probes described in the present invention are labeled with modification groups that exhibit different fluorescent colors. The primers and probes described in ID NO: 1 to ID NO: 3 and ID NO: 4 to ID NO: 6 are in one group; the primers and probes described in ID NO: 7 to ID NO: 9 and ID NO: 10 to ID NO: 12 are in one group; the primers and probes described in ID NO: 13 to ID NO: 15 and ID NO: 16 to ID NO: 18 are in one group; the primers and probes described in ID NO: 19 to ID NO: 21 and ID NO: 22 to ID NO: 24 are in one group. The above groupings constitute a dual fourplex fluorescence PCR for simultaneous detection of 8 pathogens, namely classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus type 2, African swine fever virus, swine influenza A virus, Streptococcus suis, and Pasteurella multocida.

[0033] The application described in the present invention includes constructing a dual fourplex fluorescence PCR reaction system. In the reaction system,

[0034] The concentrations of primers ID NO: 1 and ID NO: 2 are 0.25 μmol / L, and the concentration of probe ID NO: 3 is 0.1 μmol / L;

[0035] The concentrations of primers ID NO: 4 and ID NO: 5 are 0.25 μmol / L, and the concentration of probe ID NO: 6 is 0.1 μmol / L;

[0036] The concentrations of primers ID NO: 7 and ID NO: 8 are 0.25 μmol / L, and the concentration of probe ID NO: 9 is 0.1 μmol / L;

[0037] The concentrations of primers ID NO: 10 and ID NO: 11 are 0.25 μmol / L, and the concentration of probe ID NO: 12 is 0.1 μmol / L;

[0038] The concentrations of primers ID NO: 13 and ID NO: 14 are 0.25 μmol / L, and the concentration of probe ID NO: 15 is 0.20 μmol / L;

[0039] The concentrations of primers ID NO: 16 and ID NO: 17 are 0.20 μmol / L, and the concentration of probe ID NO: 18 is 0.1 μmol / L;

[0040] The concentrations of primers ID NO: 19 and ID NO: 20 are 0.25 μmol / L, and the concentration of probe ID NO: 21 is 0.1 μmol / L;

[0041] The concentrations of primers with ID NO: 22 and ID NO: 23 are 0.1 μmol / L, and the concentration of the probe with ID NO: 24 is 0.1 μmol / L.

[0042] The probes of the present invention are pairwise labeled with modification groups that exhibit different fluorescent colors.

[0043] The fluorescent reporter group of the probe for detecting classical swine fever virus shown by the probe ID NO: 3 is VIC.

[0044] The fluorescent reporter group of the probe for detecting porcine reproductive and respiratory syndrome virus shown by the probe ID NO: 6 is FAM.

[0045] The fluorescent reporter group of the probe for detecting pseudorabies virus shown by the probe ID NO: 9 is VIC.

[0046] The fluorescent reporter group of the probe for detecting porcine circovirus type 2 shown by the probe ID NO: 12 is FAM.

[0047] The fluorescent reporter group of the probe for detecting African swine fever virus shown by the probe ID NO: 15 is FAM.

[0048] The fluorescent reporter group of the probe for detecting influenza A virus of swine shown by the probe ID NO: 18 is VIC.

[0049] The fluorescent reporter group of the probe for detecting Streptococcus suis of swine shown by the probe ID NO: 21 is FAM.

[0050] The fluorescent reporter group of the probe for detecting Pasteurella multocida of swine shown by the probe ID NO: 24 is VIC.

[0051] The method steps when the present invention implements the above application include:

[0052] (1) Extract or release the nucleic acid of the sample to be tested;

[0053] (2) Preparation of the fluorescence PCR reaction system: Add the nucleic acid template of the sample to be tested, and add the dual fluorescence PCR primer-probe combination described in claims 1 to 2 to prepare a dual fourplex fluorescence PCR reaction system;

[0054] (3) Perform fluorescence PCR analysis on the nucleic acid obtained in step (1);

[0055] (4) Obtain and analyze the results.

[0056] In step (2) of the present invention, the detection reaction conditions are set as follows: reverse transcription at 55°C for 15 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 60°C for 31 s, for a total of 45 cycles; and fluorescence signals are collected at the end of each cycle.

[0057] Detection principle of this application: The most common method for etiological detection is fluorescence quantitative polymerase chain reaction (abbreviated as PCR). In the PCR reaction system, it contains a pair of specific primers and a hydrolysis probe (Taqman probe). This probe is a specific oligonucleotide sequence, with a fluorescent reporter group (5' end) and a fluorescent quenching group (3' end) labeled at both ends. When the probe is intact, the fluorescent signal emitted by the fluorescent reporter group is absorbed by the fluorescent quenching group; if there is a target sequence in the reaction system, during the PCR reaction, the probe binds to the template, and DNA polymerase uses the exonuclease activity of the enzyme to enzymatically cleave and degrade the probe, separating the fluorescent reporter group from the fluorescent quenching group, and emitting fluorescence. For each amplified DNA strand, one fluorescent molecule is generated. The fluorescence quantitative PCR instrument can monitor the cycle number (Ct value) when the fluorescence reaches the preset threshold, which is related to the nucleic acid concentration of the pathogen. The higher the nucleic acid concentration of the pathogen, the smaller the Ct value. Finally, it can be judged whether there is an infection based on the Ct value.

[0058] Compared with the prior art, the beneficial effects of the present invention are:

[0059] (1) The present invention realizes the purpose of simultaneously detecting and distinguishing 8 porcine respiratory syndrome pathogens in the same octuplet tube through one reaction, making the differential detection and diagnosis more efficient, economical, and convenient.

[0060] (2) High sensitivity: For the quadruple fluorescence PCR detection method provided by the present invention, the detection lower limits of the dual fluorescence PCR method established for plasmid standards of CSFV, PRRSV, PRV, PCV-2, ASFV, IAV-S, SS, and PM are respectively 2.096×10 1 copies / μL, 3.563×10 1 copies / μL, 1.567×10 2 copies / μL, 1.384×10 1 copies / μL, 3.698×10 2 copies / μL, 1.986×10 1 copies / μL, 2.553×10 1 copies / μL, 3.926×10 2 copies / μL, while the lowest detection lower limits of the single-reference kit method are respectively 2.096×10 1 copies / μL, 3.563×101 copies / μL, 1.567×10 3 copies / μL, 1.384×10 1 copies / μL, 3.698×10 2 copies / μL, 1.986×10 2 copies / μL, 2.553×10 1 copies / μL, 3.926×10 1 copies / μL. Comparing the two, it shows that the established duplex fluorescence PCR detection method has basically the same sensitivity as the single-reference method qPCR.

[0061] (3) Good specificity: For the duplex quadruple fluorescence PCR detection method provided by the present invention, plasmid standard products of 106 copies / μL of CSFV, PRRSV, PRV, PCV-2, ASFV, IAV-S, SS, and PM are mixed in equal proportions to prepare a 10 7 copies / μL mixture as a standard positive control, and RNase-free H2O is used as a negative control. The established duplex fluorescence PCR method is used to detect it. Only specific amplification curves and Ct values appear for the target pathogens, and there is no cross-reaction, indicating that the method has strong specificity.

[0062] (4) Fast detection speed and high throughput: For the duplex quadruple fluorescence PCR detection method provided by the present invention, the entire amplification and detection process can be completed within 1 h 30 min after adding the sample, and according to the number of detection wells of the fluorescence quantitative PCR instrument, 96-384 samples can be synchronously detected at one time.

[0063] (5) Simple operation: For the duplex quadruple fluorescence PCR detection method provided by the present invention, only the prepared reaction system needs to be placed in a fluorescence quantitative PCR instrument to complete the entire amplification and result determination process, without the need for agarose gel electrophoresis identification.

[0064] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. Description of the Drawings

[0065] Figure 1 It is the standard curve graph of the duplex fluorescence PCR of PPRSV and CSFV of the present invention. Note: The blue FAM curve is for PPRSV; the green VIC curve is for CSFV.

[0066] Figure 2 It is the curve equation graph of CSFV of the present invention.

[0067] Figure 3 It is the curve equation graph of PRRSV of the present invention.

[0068] Figure 4 This is the double fluorescence PCR standard curve graph of PRV and PCV-2 of the present invention. Note: The blue FAM curve is PCV-2; the green VIC curve is PRV.

[0069] Figure 5 This is the curve equation graph of PRV of the present invention.

[0070] Figure 6 This is the curve equation graph of PCV-2 of the present invention.

[0071] Figure 7 This is the double fluorescence PCR standard curve graph of ASFV and IAV-S of the present invention. Note: The blue FAM curve is ASFV; the green VIC curve is IAV-S.

[0072] Figure 8 This is the curve equation graph of ASFV of the present invention.

[0073] Figure 9 This is the curve equation graph of IAV-S of the present invention.

[0074] Figure 10 This is the double fluorescence PCR standard curve graph of SS and PM of the present invention. Note: The blue FAM curve is SS; the green VIC curve is PM.

[0075] Figure 11 This is the curve equation graph of SS of the present invention.

[0076] Figure 12 This is the curve equation graph of PM of the present invention.

[0077] Figure 13 This is the specific test result graph of the present invention. Note: 1: IAV-S, 2: PRV, 3: PRRSV, 4: SS, 5: CSFV, 6: PM, 7: PCV-2, 8: ASFV. Detailed implementation manners

[0078] The present invention designs and synthesizes primer and probe sequences according to the conserved genes of 8 pathogens including classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus type 2, African swine fever virus, influenza A virus of swine, Streptococcus suis, and Pasteurella multocida, and establishes a convenient and rapid application method, which can be used for the simultaneous detection of the above 8 pathogens.

[0079] In the dual quadruple fluorescence PCR primer-probe combination of the present invention, the probes are labeled with modification groups that exhibit different fluorescence colors. The primers and probes of ID NO: 1 to ID NO: 3 and ID NO: 4 to ID NO: 6 are in one group; the primers and probes of ID NO: 7 to ID NO: 9 and ID NO: 10 to ID NO: 12 are in one group; the primers and probes of ID NO: 13 to ID NO: 15 and ID NO: 16 to ID NO: 18 are in one group; the primers and probes of ID NO: 19 to ID NO: 21 and ID NO: 22 to ID NO: 24 are in one group, which constitute a dual quadruple fluorescence PCR for simultaneous detection of 8 pathogens including classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus type 2, African swine fever virus, swine influenza A virus, Streptococcus suis, and Pasteurella multocida.

[0080] Example 1:

[0081] The primers and probes used in the present invention are shown in Table 1 below:

[0082] Table 1:

[0083]

[0084] Example 2:

[0085] Preparation of positive plasmids of 8 pathogens including classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus type 2, African swine fever virus, swine influenza A virus, Streptococcus suis, and Pasteurella multocida.

[0086] The target gene sequences (sequences containing primers and probes) amplified by primers and probes of classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus type 2, African swine fever virus, swine influenza A virus, Streptococcus suis, and Pasteurella multocida were sent to Beijing Tsingke Biotechnology Co., Ltd. for synthesis and cloned into the pUC57 vector, named pUC57-KNU-1922p, pUC57-PRRSV-SDPD-2022_contig5, pUC57-PRV-gE, UC57-PCV-2, pUC57-ASFV, pUC57-IAV-S, pUC57-SS, pUC57-PM respectively. 100 μL of the bacterial liquid containing the positive plasmid was respectively added to 5 mL of LB liquid medium, 5 μL of 100 mg / mL AMP was added, and cultured overnight at 37 °C and 200 rPM in an electrothermal constant temperature incubator. The bacterial liquid containing the positive plasmid was collected, DNA standard products were extracted, the concentration of the plasmid standard products was measured using a ultra-micro nucleic acid and protein analyzer, and the copy number was calculated.

[0087] Using an ultra-micro nucleic acid and protein analyzer, the concentrations of plasmid standards of classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus type 2, African swine fever virus, influenza A virus, Streptococcus suis, and Pasteurella multocida were 61 ng / μL, 104.5 ng / μL, 45.7 ng / μL, 40.4 ng / μL, 111.1 ng / μL, 57.85 ng / μL, 74.65 ng / μL, and 119.3 ng / μL respectively. Through the formula: copy number (copies / μL) = (6.02×10 23 )×(ng / μL×10 -9 ) / (DNA length × average molecular mass), the calculated copy numbers were 2.096×10 10 copies / μL, 3.563×10 10 copies / μL, 1.567×10 10 copies / μL, 1.384×10 10 copies / μL, 3.698×10 10 copies / μL, 1.986×10 10 copies / μL, 2.553×10 10 copies / μL, 3.926×10 10 copies / μL. The plasmid standards were diluted in a 10-fold gradient and stored at -20°C for later use.

[0088] Example 3:

[0089] 3.1 Primer and probe concentration optimization and reaction program determination

[0090] 3.1.1 Singleplex fluorescence PCR primer and probe concentration optimization

[0091] The recommended amplification system (as shown in Table 2) and amplification program of HiScriptⅡU+One Step qRT-PCR Probe Kit were used for real-time amplification and detection of the 8 pathogens in this study. The concentrations of primers and probes were optimized, with three replicates in each group. The concentrations of primers and probes were optimized based on the criteria that the reaction product reached the minimum sample threshold cycle number (Ct value), the highest fluorescence value, and no non-specific amplification. The final concentrations of the upstream and downstream primers were set as: 0.1 μmol / L, 0.15 μmol / L, 0.2 μmol / L, 0.25 μmol / L, and the probe concentrations were: 0.1 μmol / L, 0.15 μmol / L, 0.2 μmol / L, 0.25 μmol / L; the amplification system was as shown in Table 2; the reaction program: reverse transcription at 55°C for 15 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for a total of 45 cycles.

[0092] Table 2:

[0093]

[0094]

[0095] Note: x represents the above different primer and probe concentrations.

[0096] 3.1.2 Optimization of annealing temperature

[0097] Prepare a dual fluorescence quantitative PCR amplification system according to the optimized primer and probe concentrations, set different annealing temperatures of 56 °C, 58 °C, and 60 °C, and perform fluorescence PCR amplification on plasmid standards of 10 3 copies / μL, 10 4 copies / μL, 10 5 copies / μL. Compare the Ct values and fluorescence intensities to determine the optimal annealing temperature.

[0098] 3.1.3 Optimization of annealing time

[0099] Prepare a dual fluorescence quantitative PCR system according to the optimized primer and probe concentrations, and set annealing times of 30 s, 31 s, and 32 s respectively. Perform fluorescence PCR amplification on plasmid standards of 10 3 -10 7 copies / μL. Compare the amplification efficiency E and R 2 of the standard curve to determine the optimal annealing time.

[0100] 3.1.4 Optimization results

[0101] By optimizing different primer and probe concentrations, calculate the average Ct value of three replicate wells. The condition with a smaller cycle number when there is a specific signal is the preferred selection factor. When approximate data appears, select the condition with high fluorescence intensity, an amplification curve that enters the plateau phase earlier, and less usage of primers and probes. Therefore, in this experiment, the final concentrations of primers and probes for dual fluorescence PCR are CSFV: 0.25 μmol / L, 0.1 μmol / L; PRRSV: 0.25 μmol / L, 0.1 μmol / L; PRV: 0.25 μmol / L, 0.1 μmol / L; PCV-2: 0.25 μmol / L, 0.1 μmol / L; ASFV: 0.25 μmol / L, 0.20 μmol / L; IAV-S: 0.20 μmol / L, 0.1 μmol / L; SS: 0.25 μmol / L, 0.1 μmol / L; PM: 0.1 μmol / L, 0.1 μmol / L.

[0102] The optimization of its amplification reaction program is shown in Table 3:

[0103] Table 3:

[0104]

[0105] Example 4:

[0106] 4.1 Plotting of the standard curve

[0107] The plasmid standard was serially diluted 10-fold, and the final plasmid concentration was diluted to 10 3 ~10 7 copies / μL at a total of 5 different concentrations. Using these as templates, dual quadruplex fluorescent PCR amplification was performed under the optimal reaction conditions. With the logarithm of the template as the x-axis and the Ct value as the y-axis, a standard curve was established.

[0108] A dual-fluorescent PCR standard curve was established (such as Figure 1 , 4 , 7, 10). For the CSFV standard curve, the correlation coefficient R2 was 0.999, the amplification efficiency E was 94.5%, and the linear equation was y = -3.4563X + 39.427 (such as Figure 2 ); for the PRSSV standard curve, the correlation coefficient R2 was 0.997, the amplification efficiency E was 100.1%, and the linear equation was y = -3.3179X + 36.814 (such as Figure 3 ); for the PRV standard curve, the correlation coefficient R2 was 0.996, the amplification efficiency E was 95.3%, and the linear equation was y = -3.4352X + 38.965 (such as Figure 5 ); for the PCV-2 standard curve, the correlation coefficient R2 was 0.993, the amplification efficiency E was 102.2%, and the linear equation was y = -3.2704X + 39.225 (such as Figure 6 ); for the ASFV standard curve, the correlation coefficient R2 was 0.996, the amplification efficiency E was 94.2%, and the linear equation was y = -3.4714X + 40.340 (such as Figure 8 ); for the IAV-S standard curve, the correlation coefficient R2 was 0.994, the amplification efficiency E was 105.4%, and the linear equation was y = -3.2016X + 37.111 (such as Figure 9 ); for the SS standard curve, the correlation coefficient R2 was 1.997, the amplification efficiency E was 109.7%, and the linear equation was y = -3.1146X + 36.152 (such as Figure 11 ); for the PM standard curve, the correlation coefficient R2 was 0.995, the amplification efficiency E was 108.2%, and the linear equation was y = -3.1359X + 38.854 (such as Figure 12 ). The amplification curves of the plasmid standards were all smooth "S"-shaped curves, showing good linear characteristics.

[0109] Example 5:

[0110] 5.1 Specificity test

[0111] Using the plasmid standard as the standard positive control and RNA-free H2O as the negative control. Nucleic acids of CSFV, PRRSV, PRV, PCV-2, ASFV, IAV-S, SS, and PM were used as templates, and amplified by the established duplex quadruple fluorescence PCR method. Observe their specific amplification curves and Ct values.

[0112] Mix 10 6 copies / μL plasmid standards of CSFV, PRRSV, PRV, PCV-2, ASFV, IAV-S, SS, and PM in equal proportions to prepare a 10 7 copies / μL mixture as the standard positive control, and RNase-free H2O as the negative control. Detect it by the established duplex fluorescence PCR method. Only the target pathogen shows specific amplification curves and Ct values, and there is no cross-reaction, indicating that this method has strong specificity (as Figure 13 ).

[0113] Example 6:

[0114] 6.1 Sensitivity test

[0115] Using plasmid standards with 4 different concentrations from 10 0 to 10 4 copies / μL as templates, respectively perform duplex quadruple fluorescence PCR amplification to detect the lowest detection limit of this method. At the same time, using plasmid standards with 4 different concentrations from 10 0 to 10 4 copies / μL as templates, respectively perform single fluorescence PCR amplification according to the method recommended in the reagent instructions to detect the lowest detection limit and compare with the established method.

[0116] The test results of the lowest detection limit are shown in Table 4. The detection limits of the established duplex fluorescence PCR method for plasmid standards of CSFV, PRRSV, PRV, PCV-2, ASFV, IAV-S, SS, and PM are 2.096×10 1 copies / μL, 3.563×10 1 copies / μL, 1.567×10 2 copies / μL, 1.384×10 1 copies / μL, 3.698×10 2 copies / μL, 1.986×10 1 copies / μL, 2.553×10 1copies / μL, 3.926×10 2 copies / μL, while the minimum detection limits of the single-reference kit method were 2.096×10 1 copies / μL, 3.563×10 1 copies / μL, 1.567×10 3 copies / μL, 1.384×10 1 copies / μL, 3.698×10 2 copies / μL, 1.986×10 2 copies / μL, 2.553×10 1 copies / μL, 3.926×10 1 copies / μL. Comparing the two, it shows that the established duplex fluorescence PCR detection method has basically the same sensitivity as the single-reference method qPCR.

[0117] Table 4:

[0118]

[0119]

[0120] Example 7:

[0121] 7.1 Repeatability test

[0122] Three different dilution concentrations (10 3 copies / μL, 10 5 copies / μL, 10 7 copies / μL) of the plasmid standard were taken for intra-group and inter-group repeat tests. Each sample was replicated 3 times, and the average Ct value (AVG), standard deviation (SD), and coefficient of variation (CV) within and between batches were calculated to analyze the experimental results.

[0123] Using 10 3 、10 5 、10 7 copies / μL of the plasmid standard as the template, each standard was made in three batches, with 3 parallel replicates in each batch. The average Ct value (AVG), standard deviation (SD), and coefficient of variation (CV) were calculated. The coefficient of variation was ≤2.5%, indicating good repeatability (as shown in Table 5).

[0124] Table 5:

[0125]

[0126]

[0127] Example 8:

[0128] 8.1 Detection of clinical samples

[0129] The established duplex quadruple fluorescence PCR method, the national standard method, the WOAH method, and the RT-qPCR method in the cited papers were used to detect 109 clinical samples simultaneously, and the coincidence rate was calculated to evaluate the applicability of the method. The formula for the coincidence rate (%) = (number of positive agreements + number of negative agreements) / (total number of samples).

[0130] Ninety-three pig fecal samples and 16 blood samples were detected using the established duplex fluorescence quantitative PCR amplification system. For PRRSV nucleic acid, 2 were positive and 107 were negative, with a positive rate of 1.83%; for IAV-S, 1 was positive and 108 were negative, with a positive rate of 0.92%; for PCV-2, 102 were positive and 7 were negative, with a positive rate of 93.6%; for SS, 52 were positive and 57 were negative, with a positive rate of 47.7%. Among them, the coincidence rate of CSFV with the national standard method (GBT36875-2018) was 100%, the coincidence rate of PRRSV with the national standard method (GBT35912-2018) was 100%, the coincidence rate of PRV with the national standard method (GBT35911-2018) was 100%, the coincidence rate of PCV-2 with the national standard method (GBT35901-2018) was 98.2%, the coincidence rate of ASFV with the national standard method (GBT18648-2020) was 100%, the coincidence rate of IAV-S with the WOAH method was 100%, and the coincidence rates of SS and PM with the paper method were 99.1% and 100% respectively (as shown in Table 6).

[0131] Table 6:

[0132]

[0133]

[0134] In summary, the primer-probe combination of the present invention and the duplex quadruple Taqman fluorescence PCR method using the primer-probe combination have high sensitivity, good specificity, are simple and rapid, and are applicable to the detection of 8 swine respiratory syndrome viruses including classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus type 2, African swine fever virus, influenza A virus of swine, Streptococcus suis, and Pasteurella multocida in China.

[0135] The foregoing description of the specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many modifications and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention, as well as various different selections and modifications. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. A biochemical kit, characterized in that, The kit includes the following primers and probes: Primer ID NO: 1, ID NO: 2 and probe ID NO: 3; Primer ID NO: 4, ID NO: 5 and probe ID NO: 6; Primer ID NO: 7, ID NO: 8 and probe ID NO: 9; Primer ID NO: 10, ID NO: 11 and probe ID NO: 12; Primer ID NO: 13, ID NO: 14 and probe ID NO: 15; Primer ID NO: 16, ID NO: 17 and probe ID NO: 18; Primer ID NO: 19, ID NO: 20 and probe ID NO: 21; Primer ID NO: 22, ID NO: 23 and probe ID NO:

24.

2. Application of the kit according to claim 1 in detecting porcine respiratory pathogens.

3. The application according to claim 2, wherein The porcine respiratory pathogens include classical swine fever virus and / or porcine reproductive and respiratory syndrome virus and / or pseudorabies virus and / or porcine circovirus type 2 and / or African swine fever virus and / or influenza A virus of swine and / or Streptococcus suis and / or Pasteurella multocida.

4. According to the application described in claim 3, wherein Primer ID NO: 1, ID NO: 2 and probe ID NO: 3 are used to detect classical swine fever virus; Primer ID NO: 4, ID NO: 5 and probe ID NO: 6 are used to detect porcine reproductive and respiratory syndrome virus; Primer ID NO: 7, ID NO: 8 and probe ID NO: 9 are used to detect pseudorabies virus; Primer ID NO: 10, ID NO: 11 and probe ID NO: 12 are used to detect porcine circovirus type 2; Primer ID NO: 13, ID NO: 14 and probe ID NO: 15 are used to detect African swine fever virus; Primer ID NO: 16, ID NO: 17 and probe ID NO: 18 are used to detect influenza A virus of swine; Primer ID NO: 19, ID NO: 20 and probe ID NO: 21 are used to detect Streptococcus suis; Primer ID NO: 22, ID NO: 23 and probe ID NO: 24 are used to detect Pasteurella multocida.

5. The application according to claims 1 and 4, characterized in that, The probes are labeled with modification groups that exhibit different fluorescent colors. The primers and probes of ID NO: 1 to ID NO: 3 and ID NO: 4 to ID NO: 6 are in one group; the primers and probes of ID NO: 7 to ID NO: 9 and ID NO: 10 to ID NO: 12 are in one group; the primers and probes of ID NO: 13 to ID NO: 15 and ID NO: 16 to ID NO: 18 are in one group; the primers and probes of ID NO: 19 to ID NO: 21 and ID NO: 22 to ID NO: 24 are in one group, which constitutes a dual fourplex fluorescence PCR for simultaneous detection of 8 pathogens including classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine circovirus type 2, African swine fever virus, influenza A virus of swine, Streptococcus suis, and Pasteurella multocida.

6. The application according to claim 4, characterized in that, The application includes constructing a dual quadruple fluorescence PCR reaction system. In the reaction system, the concentrations of primers ID NO: 1 and ID NO: 2 are 0.25 μmol / L, and the concentration of probe ID NO: 3 is 0.1 μmol / L; the concentrations of primers ID NO: 4 and ID NO: 5 are 0.25 μmol / L, and the concentration of probe ID NO: 6 is 0.1 μmol / L; the concentrations of primers ID NO: 7 and ID NO: 8 are 0.25 μmol / L, and the concentration of probe ID NO: 9 is 0.1 μmol / L; the concentrations of primers ID NO: 10 and ID NO: 11 are 0.25 μmol / L, and the concentration of probe ID NO: 12 is 0.1 μmol / L; the concentrations of primers ID NO: 13 and ID NO: 14 are 0.25 μmol / L, and the concentration of probe ID NO: 15 is 0.20 μmol / L; the concentrations of primers ID NO: 16 and ID NO: 17 are 0.20 μmol / L, and the concentration of probe ID NO: 18 is 0.1 μmol / L; the concentrations of primers ID NO: 19 and ID NO: 20 are 0.25 μmol / L, and the concentration of probe ID NO: 21 is 0.1 μmol / L; the concentrations of primers ID NO: 22 and ID NO: 23 are 0.1 μmol / L, and the concentration of probe ID NO: 24 is 0.1 μmol / L.

7. The application according to claim 5, wherein The probes are pairwise labeled with modification groups that exhibit different fluorescence colors.

8. The application according to claim 7, wherein the fluorescence reporter group of the probe for detecting classical swine fever virus shown by probe ID NO: 3 is VIC, the fluorescence reporter group of the probe for detecting porcine reproductive and respiratory syndrome virus shown by probe ID NO: 6 is FAM; the fluorescence reporter group of the probe for detecting pseudorabies virus shown by probe ID NO: 9 is VIC, the fluorescence reporter group of the probe for detecting porcine circovirus type 2 shown by probe ID NO: 12 is FAM; the fluorescence reporter group of the probe for detecting African swine fever virus shown by probe ID NO: 15 is FAM, the fluorescence reporter group of the probe for detecting influenza A virus in pigs shown by probe ID NO: 18 is VIC; the fluorescence reporter group of the probe for detecting Streptococcus suis in pigs shown by probe ID NO: 21 is FAM, the fluorescence reporter group of the probe for detecting Pasteurella multocida in pigs shown by probe ID NO: 24 is VIC.

9. The method of the application according to claim 4, wherein It includes the following steps: (1) Extract or release the nucleic acid of the sample to be tested; (2) Preparation of the fluorescence PCR reaction system: Add the nucleic acid template of the sample to be tested, and add the dual fluorescence PCR primer-probe combination described in claim 1 to prepare a dual quadruple fluorescence PCR reaction system; (3) Perform fluorescence PCR analysis on the nucleic acid obtained in step (1); (4) Obtain and analyze the results.

10. The method according to claim 8, characterized in that, In the step (2), the detection reaction conditions are set as follows: reverse transcription at 55°C for 15 min; pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 60°C for 31 s, for a total of 45 cycles; and fluorescence signals are collected at the end of each cycle.

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