CN113528708A - Triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application thereof - Google Patents

Triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application thereof Download PDF

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CN113528708A
CN113528708A CN202111036558.1A CN202111036558A CN113528708A CN 113528708 A CN113528708 A CN 113528708A CN 202111036558 A CN202111036558 A CN 202111036558A CN 113528708 A CN113528708 A CN 113528708A
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黄坚
李妍
杨晓农
郭琪
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Southwest Minzu University
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Abstract

The invention discloses a triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application thereof. The invention provides specific primers for detecting feline astrovirus, feline parvovirus and feline enterocoronavirus, and establishes a triple PCR detection method for the feline astrovirus, the feline parvovirus and the feline enterocoronavirus. The specific primer and the detection method have the advantages of strong specificity, high sensitivity, good repeatability and stability, and provide a rapid, sensitive and specific clinical detection method for differential diagnosis of the diarrhea diseases of the cat.

Description

Triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application thereof
Technical Field
The invention belongs to the field of virus nucleic acid detection, and particularly relates to a triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application thereof.
Background
Feline diarrheal diseases are multiple in clinic, among which clinical symptoms caused by infectious pathogens are the most serious, and enteroviruses, particularly represented by Feline Panleukopenia Virus (FPV), are the main causes of morbidity and mortality in young cats. In addition to FPV, viral macrogenomics studies have shown that Feline astrovirus (FAstV), Feline enteric coronavirus (FECoV), and a mixture of other enteroviruses are also present in Feline fecal samples, which may be contributing factors in determining the efficacy and prognosis of clinical antiviral therapy.
Feline Parvovirus (FPV) is also known as Feline panleukopenia virus, Feline distemper virus, or Feline infectious enteritis virus. The viral genome comprises an Open Reading Frame (ORF) encoding 4 coding genes, such as nonstructural proteins (NS) and structural proteins (VP1 and VP2), which are susceptible to recombinant variation. Clinical manifestations of cats infected with parvovirus are high fever, vomiting, diarrhea and enteritis, which can lead to a severe reduction in white blood cell counts. The virus mainly infects various animals such as felines and weasels, especially has higher morbidity and mortality rate of young animals below 6 months old, and is a virus with the widest infection range and the strongest pathogenicity in the genus of the existing carnivorous animal parvovirus.
FAstV is a non-enveloped single-stranded positive-strand RNA virus of the mammalian genus Astrovirus comprising sequences encoding the Open Reading Frames (ORFs) 1a, 1b and the 3 encoding genes of the nucleocapsid protein (N), wherein the ORF1 sequence shows significant genetic polymorphisms. This virus is evolutionarily close to the human astrovirus, and thus frequent human-cat contact in a co-occupiant environment may increase the risk of cross-infection.
FECoV is a single-stranded positive-strand RNA virus of the genus CoV, and includes 6 coding gene sequences such as ORF1a, ORF1b, spike protein (S), membrane protein (M), and nonstructural protein (N). Cats are usually free of obvious clinical symptoms following a single infection with FECoV. The infected cat still has infection risk after recovery and is recessively infected, and the virus is one of the main reasons for the continuous spread and prevalence of the virus. When the S and 3c genes of FECoV are mutated, FECoV type I may be converted into feline infectious peritonitis virus type II (FIPV) having strong pathogenicity.
At present, the molecular detection method for FPV is mature, but the clinical detection of other enteroviruses still lacks an efficient method, and at present, no molecular detection method for simultaneously detecting FPV/FAstV/FECoV exists in China. Therefore, if a triple PCR detection method capable of simultaneously detecting the infection of a plurality of feline diarrhea viruses can be developed, the method is helpful for improving the detection level of the feline diarrhea viruses and has important application prospects in the aspects of disease diagnosis, prevention and treatment and the like.
Disclosure of Invention
The invention aims to provide a triple PCR detection method for simultaneously detecting three feline diarrhea viruses, which can simultaneously detect feline astrovirus (FAstV), Feline Parvovirus (FPV) and feline enterocoronavirus (FECoV).
The purpose of the invention is realized by the following scheme:
provides a primer combination for simultaneously detecting three feline diarrhea viruses, which comprises detection primers of FAstV, FPV and FECoV, and the specific primer sequences are as follows:
FAstV-F：5’-CTGGAAGAGCTATGACACCATTG-3’，
FAstV-R：5’-CCATGAACGCAGAGAGCAAC-3’；
FPV-F：5’-TGGTTCTGGGGGTGTGGG-3’，
FPV-R：5’-GCTGCTGGAGTAAATGGC-3’；
FECoV-F：5’-CCAAAAGACGTGGTCGTTCTAA-3’；
FECoV-R：5’-GTTTTCTTCCAGGTGTGTTTGT-3’。
the pathogen of the virus causing the diarrhea of the cat in clinic is various, but the common pathogen is still FPV, the condition that the FAstV and the FECoV directly or indirectly cause the clinical morbidity is not paid enough attention, one of the reasons is that an efficient detection method is not provided, the primer combination provided by the invention can be used for simultaneously detecting the three viruses, so that the multiple pathogen detection on the same sample is avoided, and the detection efficiency is improved.
The primer sequence of the FAstV is selected from an open reading frame encoding ORF1b, the primer sequence of the FPV is selected from a virus protein VP2 gene sequence, and the primer sequence of the FECoV is selected from an open reading frame encoding ORF1 b.
The invention also provides a triple PCR detection method for simultaneously detecting three feline diarrhea viruses, which is characterized in that the primer combination is used for simultaneously detecting the FAstV, the FPV and the FECoV;
the working concentration ratio of each primer in the primer combination pair is as follows: FAstV: FPV (field programmable volume): FECoV is 0.8-1.4: 0.8-1.4: 0.8 to 1.4.
Further, the working concentration ratio of each primer in the primer combination pair is: FAstV: FPV (field programmable volume): FECoV is 0.8-1.2: 0.8: 0.8 to 1.2.
The reaction system of the triple PCR is as follows:
0.8-1.5 mu L of mixed template DNA of FAstV, FPV and FECoV, 2 xTaq PCR Master Mix 10-15 mu L, 0.3-0.8 mu mol/L of upstream and downstream primers of FAstV, 0.3-0.8 mu mol/L of upstream and downstream primers of FPV, 0.3-0.8 mu mol/L of upstream and downstream primers of FECoV, and ddH2Make up to 25. mu.L of O.
Further, the reaction system of the triple PCR is as follows:
1.5. mu.L of mixed template DNA of FAstV, FPV and FECoV, 12.5. mu.L of 2 XTAQ PCR Master Mix, 0.5. mu. mol/L of both the upstream and downstream primers of FAstV, 0.4. mu. mol/L of both the upstream and downstream primers of FPV, and 0.6. mu. mol/L of both the upstream and downstream primers of FECoV, using ddH2Make up to 25. mu.L of O.
The amplification procedure of the triple PCR is as follows:
pre-denaturation at 92-97 ℃ for 1.5-2.5 min, at 90-97 ℃ for 15-40 s, at 45-60 ℃ for 15-40 s, at 70-75 ℃ for 20-35 s, for 30-35 cycles, and finally extending at 70-75 ℃ for 8-15 min;
further, the amplification procedure of the triple PCR is as follows: pre-denaturation at 95 ℃ for 2min, 30s at 95 ℃, 30s at 50-54 ℃ and 30s at 72 ℃ for 35 cycles, and finally extension at 72 ℃ for 10 min.
The invention also provides application of the primer combination in preparing a kit for detecting the feline astrovirus, the feline parvovirus and the feline enterocoronavirus.
Further, in the kit, the working concentration ratio of each primer in the primer combination pair is as follows: FAstV: FPV (field programmable volume): FECoV is 0.8-1.4: 0.8-1.4: 0.8 to 1.4.
Further, in the kit, the working concentration of each primer is as follows: the primers for both the upstream and downstream of the FAstV were 0.5. mu. mol/L, for the FPV 0.4. mu. mol/L, and for the FECoV 0.6. mu. mol/L.
Further, the reaction procedures of the kit in work are as follows: pre-denaturation at 95 ℃ for 2min, 30s at 95 ℃, 30s at 50-54 ℃ and 30s at 72 ℃ for 35 cycles, and finally extension at 72 ℃ for 10 min.
The invention has the following beneficial effects:
(1) the primer combination can simultaneously detect the feline astrovirus (FAstV), the Feline Parvovirus (FPV) and the feline enterocoronavirus (FECoV).
(2) The detection method provided by the invention has the advantages of high detection efficiency, good specificity, sensitivity, repeatability and stability, and the lowest detection limit is 2.4-7.1 pg/mu L, so that an important technical means is provided for the rapid diagnosis of the diarrhea diseases of the clinical cats and the epidemiological investigation.
Drawings
FIG. 1 is an electrophoretogram of results of an orthogonal assay of three viral primer concentrations;
FIG. 2 is an electrophoretogram of the results of optimization of annealing temperatures for triple PCR reactions;
FIG. 3 is an electrophoresis chart showing the results of a triple PCR specificity test;
FIG. 4 is an electrophoretogram showing the results of triple PCR sensitivity test;
FIG. 5 is an electrophoretogram of the triple PCR amplification result of the sample.
Wherein, in fig. 1, 1-36: a sample; n: negative control;
in FIG. 2, 1-6: 48 ℃, 50 ℃, 52 ℃, 54 ℃, 56 ℃ and 58 ℃; n: negative control;
in FIG. 3, 1-3: FPV, FAstV and FECoV; 4: mixed plasmids of FPV, famtv and FECoV; 5-9: FRV, FCV, FNoV, CPV, and CCoV; n: negative control
In FIG. 4, 1-8: 100、101、102、103、104、105、106、107Gradient dilution; n: negative control;
in FIG. 5, 1-22: a portion of a clinical sample; n: negative control; p: mixing the plasmid templates; m in FIGS. 1 to 5: and (5) DNA Marker II.
Detailed Description
The technical solutions of the present invention are described clearly and completely below, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Positive samples of Feline astrovirus (FAstV), Feline Parvovirus (FPV), Feline enterocoronavirus (FECoV), Feline Norovirus (Feline Norvirus, FNoV), Feline Rhinotracheitis Virus (FRV), Feline Calicivirus (FCV), Canine Parvovirus (CPV), and Canine coronavirus (CCoV) were identified and stored by the animal medicine laboratory at the university of Western southern ethnicity. 207 cat stool samples (66 clinical healthy samples, 141 diarrhea samples) were collected from 5 pet hospitals in the metropolitan city (11 months 11-2020 months 2019).
The virus genome DNA/RNA extraction kit, Escherichia coli DH5 alpha competent cells, DNA Marker II and 2 XTaq Mastermix are purchased from Tiangen Biochemical technology (Beijing) Co., Ltd; RNAioso Plus, pMD19-T vector, Prime ScriptTMRT is purchased from TaKaRa Bio-engineering (Dalian) products Ltd; the E.Z.N.A. Gel Extraction Kit Gel recovery Kit and the E.Z.N.A.TM.plasmid Kit Plasmid Extraction Kit were purchased from OMEGA.
The test methods used in the following examples are all conventional methods unless otherwise specified. Materials and reagents used in the following examples are commercially available unless otherwise specified.
The primer sequence of the FAstV is selected from an open reading frame encoding ORF1b, the primer sequence of the FPV is selected from a virus protein VP2 gene sequence, and the primer sequence of the FECoV is selected from an open reading frame encoding ORF1 b.
Example 1
The 3 cat diarrhea virus primer sequences designed by the invention are synthesized by Shanghai biological engineering Co., Ltd, and the primer information is shown in Table 1.
TABLE 1 primer information
Figure BDA0003247228480000051
Example 2 establishment of triple PCR detection method for three Cat viruses
1. Preparation of plasmid standard substance and extraction of plasmid DNA
Taking positive samples of FAstV, FPV and FECoV, extracting nucleic acid or reversely transcribing the nucleic acid into cDNA according to the instruction of a DNA or RNA extraction kit, and respectively amplifying target gene segments. And purifying, recovering and connecting the positive PCR product to a pMD19-T vector to construct three recombinant plasmids of pMD19-T-FAstV ORF1b, pMD19-T-FPV VP2 and pMD19-T-FECoV N, respectively transforming escherichia coli DH5 alpha competent cells, screening out positive clones, and sending the positive clones to a company Limited in bioengineering (Shanghai) biology engineering for sequencing and identification.
And (3) shaking and propagating the positive bacteria with correct sequencing, extracting plasmid DNA according to the specification of the E.Z.N.A.TM. Plassmid Kit plasmid extraction Kit, and measuring the concentration of the eluted plasmid DNA by using a nucleic acid concentration measuring instrument.
Establishment and condition optimization of triple PCR of FAstV, FPV and FECoV
Diluting the extracted FPV, FAstV and FECoV plasmid DNA to the same order of magnitude, respectively taking 20 mu L of the diluted FPV, FAstV and FECoV plasmid DNA, uniformly mixing the diluted FPV, FAstV and FECoV plasmid DNA to serve as a template of triple PCR, and optimizing the final concentration of a primer and the annealing temperature of a reaction system.
The primer concentration optimized PCR total reaction system is 25 mu L: the three primer pairs of FAstV, FPV and FECoV were mixed in proportion to the final concentration shown in Table 2, and then ddH was used to Mix 1.5. mu.L of mixed template DNA of FAstV, FPV and FECoV, 2 XTAQ PCR Master Mix 12.5. mu.L, and FAstV, FPV and FECoV2And O is used for complementing the reaction system.
The annealing temperature optimized total reaction system was 25 μ L: mixed template DNA of FAstV, FPV and FECoV 1.5. mu.L; 2 × Taq PCR Master Mix 12.5 μ L; adding upstream and downstream primers under optimized concentration conditions, and finally adding ddH2And O is used for complementing the reaction system.
The amplification procedure was: pre-denaturation at 95 deg.C for 2min, 30s at 95 deg.C, 30s at 52 deg.C, 30s at 72 deg.C for 35 cycles, and final extension at 72 deg.C for 10 min; the annealing temperature in the amplification procedure was set to 6 different gradients of 48 ℃, 50 ℃, 52 ℃, 54 ℃, 56 ℃ and 58 ℃ respectively, and negative controls were set. The PCR amplification products were identified by electrophoresis on a 1.2% agarose gel.
After the amplification, 8. mu.L of the PCR product was subjected to electrophoresis on 1.2% agarose gel to determine the optimal primer concentration.
TABLE 2 primer concentration orthogonal screening for triple PCR
Figure BDA0003247228480000061
And (3) condition optimization results:
the three pairs of primers can amplify corresponding virus target genes, the electrophoresis result and the sequencing result are consistent with the reference gene, the primer concentration optimization test result is shown in figure 1, and the annealing temperature optimization test result is shown in figure 2.
Finally determining a 25 mu L reaction system of the triple PCR by optimizing conditions such as primer concentration, annealing temperature and the like: the upstream and downstream primers of the FAstV, FPV and FECoV are respectively 0.5 muL, 0.4 muL and 0.6 muL; 2 × Taq MasterMix 12.5 μ L; template 1.5 μ L; ddH2O8. mu.L, annealing temperature 54 ℃.
The target fragments with the sizes of FPV 468bp, FAstV 320bp and FECoV 664bp are respectively amplified by the multiplex PCR, and all the amplified specific target fragments are verified by sequencing.
Example 3 testing of triple PCR method
(1) Specificity test
And respectively detecting a mixed template of FAstV/FPV/FECoV, a single template of FAstV, FPV and FECoV and nucleic acids of FNoV, FRV, FCV, CPV and CCoV as templates by using an optimized triple PCR method to identify the specificity of the method.
As can be seen from the results in FIG. 3, except FPV, FAstV and FECoV, no other pathogen can amplify the corresponding band, indicating that the multiplex PCR method established by the method of the present invention has good specificity.
(2) Sensitivity test
The obtained three kinds of plasmid DNA templatesThe plates were each diluted 10-fold in gradient, for a total of 8 gradients, 1X 10 each0、1×101、1×102、1×103、1×104、1×105、1×106、1×107(ii) a 0.5 mu L of DNA is respectively taken from each dilution gradient and mixed evenly to be used as a triple PCR template, and the optimized triple PCR is used for amplification to detect the sensitivity of the method.
As can be seen from the results in FIG. 4, the lowest limit of the multiplex PCR method established by the present invention for detecting FPV, FAstV and FECoV is 2.4pg/μ L, 7.1pg/μ L and 5.1pg/μ L, respectively.
(3) Repeatability test
The established triple PCR method was used to perform 4-lot duplicate experiments on the mixed nucleic acid samples of FAstV/FPV/FECoV to verify the stability of triple PCR.
The established multiplex PCR method is used for repeatedly detecting the target template in 4 batches, and the test result is consistent and stable, which shows that the method has good repeatability.
(4) Clinical application
The established triple PCR method is used for detecting FAstV, FPV and FECoV on 207 cat stool samples, and the detection result is compared with the detection result of the single PCR.
The virus molecule detection is carried out on 207 cat anus swab samples by adopting the multiplex PCR method and the single PCR method established by the invention.
The detection result of the multiple PCR method shows that the detection rate of the FAstV is 24.15 percent (50/207), wherein the detection rate of the diarrhea cats is 24.8 percent (35/141) which is slightly higher than that of the clinical healthy cats (15/66); the detection rate of FPV was 37.20% (77/207), with 48.9% (69/141) significantly higher in diarrhea cats than in clinical healthy cats 12.1% (8/66); the detection rate of FECoV was 15.46% (32/207), with 12.7% (20/141) lower in diarrhea cats than in clinical healthy cats (12/66). The results of amplification of a portion of the clinical samples are shown in FIG. 5. Among them, the rates of mixed infection of FPV/FAstV, FPV/FECoV and FAstV/FECoV were 9.18% (19/207), 6.28% (13/207) and 6.28% (13/207), respectively, and triple mixed infection of FPV/FAstV/FECoV was less frequent (2.42%, 5/207). The conformity of the detection result and the single PCR result is 100%, which shows that the method of the invention can rapidly detect the three pathogens.
In conclusion, the invention obtains the triple PCR detection method with good specificity, sensitivity and stability by condition optimization, and the minimum detection limit obtained after gradient dilution of the template is 2.4-7.1 pg/mu L. Through the detection of clinical samples, the consistency of the positive detection of the triple PCR and the single PCR is verified, and the rechecking rate is 100 percent, which shows that the repeatability is good. Because DNA virus and RNA virus have difference in the process of extracting nucleic acid and amplification, when the detection method is established, the conditions of primer concentration, annealing temperature and the like of amplification of the 3 viruses are repeatedly screened and optimized through orthogonal tests, the triple PCR method of the FPV, the FAstV and the FECoV, which is established by the invention, takes account of different segment sizes and mismatched recombination conditions of the primers and the amplification efficiency of different viruses in a reaction system, and provides a rapid and accurate method for clinical detection of related pathogens.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
<110> university of southwest ethnic group
<120> triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application thereof
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Claims (10)

1. A primer combination for simultaneously detecting three feline diarrhea viruses is characterized by comprising detection primers of FAstV, FPV and FECoV, wherein specific primer sequences are as follows:
FAstV-F：5’-CTGGAAGAGCTATGACACCATTG-3’，
FAstV-R：5’-CCATGAACGCAGAGAGCAAC-3’；
FPV-F：5’-TGGTTCTGGGGGTGTGGG-3’，
FPV-R：5’-GCTGCTGGAGTAAATGGC-3’；
FECoV-F：5’-CCAAAAGACGTGGTCGTTCTAA-3’；
FECoV-R：5’-GTTTTCTTCCAGGTGTGTTTGT-3’。
2. a triple PCR detection method for simultaneously detecting three feline diarrhea viruses, which is characterized in that the primer combination of claim 1 is used for simultaneously detecting FAstV, FPV and FECoV;
the working concentration ratio of each primer in the primer combination pair is as follows: FAstV: FPV (field programmable volume): FECoV is 0.8-1.4: 0.8-1.4: 0.8 to 1.4.
3. The triple PCR detection method of claim 2, wherein the working concentration ratio of each primer in the primer combination pair is: FAstV: FPV (field programmable volume): FECoV is 0.8-1.2: 0.8: 0.8 to 1.2.
4. The triple PCR detection method according to claim 2, wherein the reaction system of the triple PCR is:
0.8-1.5 mu L of mixed template DNA of FAstV, FPV and FECoV, 10-15 mu L of 2 xTaq PCR Master Mix, 0.3-0.8 mu mol/L of upstream and downstream primers of FAstV, 0.3-0.8 mu mol/L of upstream and downstream primers of FPV, 0.3-0.8 mu mol/L of upstream and downstream primers of FECoV, and ddH2Make up to 25. mu.L of O.
5. The triple PCR detection method according to claim 4, wherein the reaction system of the triple PCR is:
1.5. mu.L of mixed template DNA of FAstV, FPV and FECoV, 12.5. mu.L of 2 XTAQ PCR Master Mix, 0.5. mu. mol/L of both the upstream and downstream primers of FAstV, 0.4. mu. mol/L of both the upstream and downstream primers of FPV, and 0.6. mu. mol/L of both the upstream and downstream primers of FECoV, using ddH2Make up to 25. mu.L of O.
6. The triple PCR detection method according to claim 2, wherein the amplification procedure of the triple PCR is as follows:
pre-denaturation at 92-97 ℃ for 1.5-2.5 min, at 90-97 ℃ for 15-40 s, at 45-60 ℃ for 15-40 s, at 70-75 ℃ for 20-35 s, for 30-35 cycles, and finally extending at 70-75 ℃ for 8-15 min;
further, the amplification procedure of the triple PCR is as follows: pre-denaturation at 95 ℃ for 2min, 30s at 95 ℃, 30s at 50-54 ℃ and 30s at 72 ℃ for 35 cycles, and finally extension at 72 ℃ for 10 min.
7. Use of the primer combination of claim 1 for the preparation of a kit for the detection of feline astrovirus, feline parvovirus, and feline enterocoronavirus.
8. The use according to claim 7, wherein in the kit, the working concentration ratio of each primer in the primer combination pair is: FAstV: FPV (field programmable volume): FECoV is 0.8-1.4: 0.8-1.4: 0.8 to 1.4.
9. The use according to claim 7, wherein in the kit, the working concentration of each primer is: the primers for both the upstream and downstream of the FAstV were 0.5. mu. mol/L, for the FPV 0.4. mu. mol/L, and for the FECoV 0.6. mu. mol/L.
10. The use according to claim 7, wherein the reaction program of the kit in operation is: pre-denaturation at 95 ℃ for 2min, 30s at 95 ℃, 30s at 50-54 ℃ and 30s at 72 ℃ for 35 cycles, and finally extension at 72 ℃ for 10 min.
CN202111036558.1A 2021-09-06 2021-09-06 Triple PCR detection method for simultaneously detecting three feline diarrhea viruses and application thereof Pending CN113528708A (en)

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CN115852043A (en) * 2022-07-19 2023-03-28 安徽农业大学 Multiplex fluorescence PCR primer probe group for detecting four cat diarrhea-related viruses, kit and application
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Cited By (6)

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Publication number Priority date Publication date Assignee Title
CN112921122A (en) * 2021-03-26 2021-06-08 广西大学 Multiplex PCR (polymerase chain reaction) rapid detection kit for common feline viruses and primer group thereof
CN115852043A (en) * 2022-07-19 2023-03-28 安徽农业大学 Multiplex fluorescence PCR primer probe group for detecting four cat diarrhea-related viruses, kit and application
CN116875743A (en) * 2023-09-07 2023-10-13 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Fluorescent quantitative PCR kit for detecting two cat enteroviruses at one time and application thereof
CN116875743B (en) * 2023-09-07 2023-12-29 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Fluorescent quantitative PCR kit for detecting two cat enteroviruses at one time and application thereof
CN116949224A (en) * 2023-09-21 2023-10-27 上海基灵生物科技有限公司 Multiplex PCR (polymerase chain reaction) kit for detecting pathogens in cat digestive tract and application thereof
CN116949224B (en) * 2023-09-21 2023-12-15 上海基灵生物科技有限公司 Multiplex PCR (polymerase chain reaction) kit for detecting pathogens in cat digestive tract and application thereof

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Application publication date: 20211022