CN117844956A - Fluorescent PCR primer probe set, kit and method for quantitatively detecting haemophilus parasuis - Google Patents
Fluorescent PCR primer probe set, kit and method for quantitatively detecting haemophilus parasuis Download PDFInfo
- Publication number
- CN117844956A CN117844956A CN202410251918.7A CN202410251918A CN117844956A CN 117844956 A CN117844956 A CN 117844956A CN 202410251918 A CN202410251918 A CN 202410251918A CN 117844956 A CN117844956 A CN 117844956A
- Authority
- CN
- China
- Prior art keywords
- concentration
- fluorescent pcr
- fluorescent
- detection
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000523 sample Substances 0.000 title claims abstract description 84
- 241000606807 Glaesserella parasuis Species 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 42
- 238000001514 detection method Methods 0.000 claims abstract description 104
- 230000003321 amplification Effects 0.000 claims description 71
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 71
- 108010006785 Taq Polymerase Proteins 0.000 claims description 21
- 102000004190 Enzymes Human genes 0.000 claims description 20
- 108090000790 Enzymes Proteins 0.000 claims description 20
- 229940088598 enzyme Drugs 0.000 claims description 20
- 239000013612 plasmid Substances 0.000 claims description 18
- 201000010099 disease Diseases 0.000 claims description 15
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- 238000002474 experimental method Methods 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 8
- 239000013641 positive control Substances 0.000 claims description 8
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 125000006853 reporter group Chemical group 0.000 claims description 6
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 238000010791 quenching Methods 0.000 claims description 4
- 230000000171 quenching effect Effects 0.000 claims description 4
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 4
- 241000283690 Bos taurus Species 0.000 claims description 3
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 claims description 3
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 3
- 108090000190 Thrombin Proteins 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 108091008324 binding proteins Proteins 0.000 claims description 3
- 239000003161 ribonuclease inhibitor Substances 0.000 claims description 3
- 229960004072 thrombin Drugs 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 2
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 2
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 claims description 2
- 229940035893 uracil Drugs 0.000 claims description 2
- 102000014914 Carrier Proteins Human genes 0.000 claims 1
- 235000011187 glycerol Nutrition 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 abstract description 6
- 230000006872 improvement Effects 0.000 abstract description 2
- 238000011529 RT qPCR Methods 0.000 abstract 1
- 238000003753 real-time PCR Methods 0.000 description 31
- 238000006243 chemical reaction Methods 0.000 description 24
- 241000282898 Sus scrofa Species 0.000 description 22
- 238000000137 annealing Methods 0.000 description 18
- 229940031551 inactivated vaccine Drugs 0.000 description 16
- 238000004519 manufacturing process Methods 0.000 description 14
- 239000003153 chemical reaction reagent Substances 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- 238000012360 testing method Methods 0.000 description 11
- 238000012795 verification Methods 0.000 description 10
- 238000012408 PCR amplification Methods 0.000 description 9
- 229960005486 vaccine Drugs 0.000 description 9
- 108020004707 nucleic acids Proteins 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 230000008569 process Effects 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 201000008283 Atrophic Rhinitis Diseases 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- 241000702619 Porcine parvovirus Species 0.000 description 6
- 206010039088 Rhinitis atrophic Diseases 0.000 description 6
- 241000194021 Streptococcus suis Species 0.000 description 6
- 241000282887 Suidae Species 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 208000024949 Swine Erysipelas Diseases 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 241000606748 Actinobacillus pleuropneumoniae Species 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000204045 Mycoplasma hyopneumoniae Species 0.000 description 4
- 241001673669 Porcine circovirus 2 Species 0.000 description 4
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 4
- 102100037111 Uracil-DNA glycosylase Human genes 0.000 description 4
- 230000002458 infectious effect Effects 0.000 description 4
- 238000002844 melting Methods 0.000 description 4
- 230000008018 melting Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 241000193171 Clostridium butyricum Species 0.000 description 3
- 241000194032 Enterococcus faecalis Species 0.000 description 3
- 102100038353 Gremlin-2 Human genes 0.000 description 3
- 102000015728 Mucins Human genes 0.000 description 3
- 108010063954 Mucins Proteins 0.000 description 3
- 101001032860 Mus musculus Gremlin-2 Proteins 0.000 description 3
- 206010035664 Pneumonia Diseases 0.000 description 3
- 241000241413 Propolis Species 0.000 description 3
- 241000607142 Salmonella Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229940032049 enterococcus faecalis Drugs 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 210000002751 lymph Anatomy 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 229940069949 propolis Drugs 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 238000012270 DNA recombination Methods 0.000 description 2
- 241000204031 Mycoplasma Species 0.000 description 2
- 241000606856 Pasteurella multocida Species 0.000 description 2
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 2
- 206010058556 Serositis Diseases 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- WOLHOYHSEKDWQH-UHFFFAOYSA-N amantadine hydrochloride Chemical compound [Cl-].C1C(C2)CC3CC2CC1([NH3+])C3 WOLHOYHSEKDWQH-UHFFFAOYSA-N 0.000 description 2
- 229960001280 amantadine hydrochloride Drugs 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- 206010003246 arthritis Diseases 0.000 description 2
- 102000023732 binding proteins Human genes 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 229940051027 pasteurella multocida Drugs 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 201000006509 pleuropneumonia Diseases 0.000 description 2
- 239000006041 probiotic Substances 0.000 description 2
- 235000018291 probiotics Nutrition 0.000 description 2
- 208000009305 pseudorabies Diseases 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 229960000329 ribavirin Drugs 0.000 description 2
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- AYIRNRDRBQJXIF-NXEZZACHSA-N (-)-Florfenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CF)NC(=O)C(Cl)Cl)C=C1 AYIRNRDRBQJXIF-NXEZZACHSA-N 0.000 description 1
- RDEIXVOBVLKYNT-VQBXQJRRSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(1-aminoethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2-yl]o Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)C(C)N)N)[C@@H](N)C[C@H]1N.O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-VQBXQJRRSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- PTNZGHXUZDHMIQ-UHFFFAOYSA-N 4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide;hydrochloride Chemical compound Cl.C1=CC=C2C(C)C(C(O)C3C(C(O)=C(C(N)=O)C(=O)C3N(C)C)(O)C3=O)C3=C(O)C2=C1O PTNZGHXUZDHMIQ-UHFFFAOYSA-N 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- RFLHUYUQCKHUKS-JUODUXDSSA-M Ceftiofur sodium Chemical compound [Na+].S([C@@H]1[C@@H](C(N1C=1C([O-])=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1CSC(=O)C1=CC=CO1 RFLHUYUQCKHUKS-JUODUXDSSA-M 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 241000186810 Erysipelothrix rhusiopathiae Species 0.000 description 1
- 241000606790 Haemophilus Species 0.000 description 1
- 206010023232 Joint swelling Diseases 0.000 description 1
- 241001514666 Kunzea Species 0.000 description 1
- VHJLVAABSRFDPM-IMJSIDKUSA-N L-1,4-dithiothreitol Chemical compound SC[C@H](O)[C@@H](O)CS VHJLVAABSRFDPM-IMJSIDKUSA-N 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 241000606860 Pasteurella Species 0.000 description 1
- 206010034107 Pasteurella infections Diseases 0.000 description 1
- FPVRUILUEYSIMD-RPRRAYFGSA-N [(8s,9r,10s,11s,13s,14s,16r,17r)-9-fluoro-11-hydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-3-oxo-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-17-yl] acetate Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(OC(C)=O)[C@@]1(C)C[C@@H]2O FPVRUILUEYSIMD-RPRRAYFGSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 229960003022 amoxicillin Drugs 0.000 description 1
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229960004467 ceftiofur sodium Drugs 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 150000003841 chloride salts Chemical group 0.000 description 1
- 210000004081 cilia Anatomy 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229960003657 dexamethasone acetate Drugs 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229960004082 doxycycline hydrochloride Drugs 0.000 description 1
- YXQXDXAHCSEVSD-GCYNEOGWSA-N dynamutilin Chemical compound OC(=O)\C=C\C(O)=O.CCN(CC)CCSCC(=O)O[C@@H]1C[C@@](C)(C=C)[C@@H](O)[C@H](C)[C@@]23CC[C@@H](C)[C@]1(C)[C@@H]2C(=O)CC3 YXQXDXAHCSEVSD-GCYNEOGWSA-N 0.000 description 1
- 229960003760 florfenicol Drugs 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000001046 green dye Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000010871 livestock manure Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229940051875 mucins Drugs 0.000 description 1
- 230000000474 nursing effect Effects 0.000 description 1
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 1
- 201000005115 pasteurellosis Diseases 0.000 description 1
- 238000009304 pastoral farming Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 229940092292 tiamulin fumarate Drugs 0.000 description 1
- 229960000223 tilmicosin Drugs 0.000 description 1
- JTSDBFGMPLKDCD-XVFHVFLVSA-N tilmicosin Chemical compound O([C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CCN1C[C@H](C)C[C@H](C)C1)CO[C@H]1[C@@H]([C@H](OC)[C@H](O)[C@@H](C)O1)OC)CC)[C@@H]1O[C@H](C)[C@@H](O)[C@H](N(C)C)[C@H]1O JTSDBFGMPLKDCD-XVFHVFLVSA-N 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of bacterial detection, and particularly discloses a fluorescent PCR primer probe set, a kit and a method for quantitatively detecting haemophilus parasuis, wherein the fluorescent PCR primer probe set comprises a set of primer pairs and a specific probe shown as SEQ ID No. 5; and the primer pair is selected from a first primer pair shown as SEQ ID No. 1 and SEQ ID No. 2 or a second primer pair shown as SEQ ID No. 3 and SEQ ID No. 4. Compared with the traditional qPCR detection, the method has higher positive detection rate, and has obvious improvement in detection time and pollution resistance. Provides reliable technical guarantee for rapidly and accurately realizing the detection of haemophilus parasuis.
Description
Technical Field
The invention relates to the technical field of bacterial detection, in particular to a fluorescent PCR primer probe set, a kit and a method for quantitatively detecting haemophilus parasuis.
Background
Haemophilus parasuis disease is also known as multiple cellulose serositis and arthritis, also known as glazer's disease. The disease is caused by haemophilus parasuis (Haemophilus parasuis, HPS), a bacterium that is ubiquitous in the environment. The bacteria can be extracted from both healthy and diseased swine herds. The disease is an infectious disease characterized clinically by elevated body temperature, joint swelling, dyspnea, multiple serositis, arthritis and high mortality, severely jeopardizing the health of piglets and young pigs. Pigs are the only host of the disease, the disease can affect pigs of 2 to 4 months of age, and is mainly developed before and after weaning and in the nursing stage, usually seen in pigs of 5 to 8 weeks of age, the incidence rate is generally 10 to 15 percent, and the mortality rate can reach 50 percent in severe cases, so that the disease is usually first developed for pigs with good fatness. The sick pigs can expel toxin through the respiratory system, and the disease is more likely to occur when reproductive respiratory syndrome, influenza or local pneumonia exist in pig groups. The conditions of bad living environment, water break, weaning, group transfer, group mixing, transportation and the like are also one of the causes affecting the occurrence of the disease. Haemophilus parasuis disease has affected the development of the pig industry worldwide, bringing great economic losses to the pig industry.
Because the bacteria are often mixed with other bacteria and viruses, and the cross protection force among serotypes is weak, the vaccine immunization effect is poor, so that the disease is popular in a large area; and no effective treatment method exists at present, which has a great influence on the pig industry. The real-time fluorescence quantitative PCR (qPCR) method is the most commonly used method in HPS detection at present due to simple operation and rapid detection. Meanwhile, in order to meet the market demand of HPS detection, some enterprises develop commercial kits for HPS detection using fluorescent quantitative PCR technology. At present, the design of primer probes of the fluorescent quantitative PCR detection method for haemophilus parasuis is mostly selected on a bacterial 16s rDNA sequence, the differentiation of other bacteria of the genus haemophilus of the family Pasteureidae is not high, the detection specificity is poor, the minimum detection limit of the bacteria is 100 copies/mu L or more, and the sensitivity is low. Meanwhile, the reagents have the defects of complex operation process, long detection time, low accuracy, poor anti-pollution capability and the like to a certain extent, and the requirements of HPS nucleic acid detection are hardly met.
In order to better meet the requirements of rapid and early-stage accurate detection of HPS, an ultra-rapid and high-precision qPCR detection method is developed and established, so that the purposes of effectively preventing and blocking the transmission of HPS, controlling epidemic propagation and promoting the healthy development of pig industry are achieved.
Disclosure of Invention
Aiming at the prior art, the invention aims to overcome the defects that the detection reagent for haemophilus parasuis has complex operation process, long detection time, low accuracy, poor anti-pollution capability and the like to a certain extent in the prior art, and hardly meets the requirement of HPS nucleic acid detection, thereby providing a fluorescent PCR primer probe set, a kit and a method for quantitatively detecting haemophilus parasuis, which can rapidly, sensitively and accurately realize the detection of haemophilus parasuis.
In order to achieve the aim, the invention provides a fluorescent PCR primer probe group for quantitatively detecting haemophilus parasuis, which comprises a group of primer pairs and a specific probe shown as SEQ ID No. 5; and, in addition, the method comprises the steps of,
the primer pair is selected from a first primer pair shown as SEQ ID No. 1 and SEQ ID No. 2 or a second primer pair shown as SEQ ID No. 3 and SEQ ID No. 4.
Further, in order to obtain a higher fluorescence value and a smaller Ct value to further improve the accuracy of detection, the primer pair is preferably the first primer pair shown as SEQ ID No. 1 and SEQ ID No. 2.
Preferably, the 5' end of the specific probe is marked with a fluorescent reporter group;
and/or, the 3' -end of the specific probe is marked with a fluorescence quenching group.
Preferably, in a specific embodiment, the fluorescent reporter group is selected from one of 6-FAM, HEX, TET, cy, cy5, cy5.5, texas Red, yakima Yellow and VIC;
and/or the fluorescence quenching group is selected from TAMRA, MGB, BHQ-1, BHQ-2 and BHQ-3.
Preferably, the fluorescence reporter group is VIC and the fluorescence quencher group is TAMRA.
The invention also provides a fluorescent PCR detection kit for quantitatively detecting haemophilus parasuis, which comprises the fluorescent PCR primer probe set and the hot start Taq DNA polymerase premix.
Preferably, the hot start Taq DNA polymerase premix at least comprises a PCR buffer and an enzyme mixture containing hot start Taq enzyme. The PCR buffer and enzyme mixture herein may be formulated using a formulation that will be understood and used by those skilled in the art.
In addition, it is particularly necessary to propose that in the fluorescent PCR detection kit of the present invention, the fluorescent PCR primer probe set and the hot-start Taq DNA polymerase premix exist in the kit in a fully premixed form. In the actual detection, the DNA template to be detected is only required to be directly subjected to fluorescent PCR reaction by adopting a kit with full premix (comprising fluorescent PCR primer probe group and hot start Taq DNA polymerase premix), and the primer and each component in the hot start Taq DNA polymerase premix are not required to be sequentially put in sequence. Based on the above, the fluorescent PCR detection kit greatly simplifies the operation steps.
Preferably, in the fluorescent PCR detection kit, the concentration of each primer in the primer pair is 0.20-0.40. Mu. Mol/L independently, and the concentration of the specific probe is 0.15-0.25. Mu. Mol/L.
In the actual operation process, the whole reaction process is as follows: the DNA template is added into the fluorescent PCR detection kit to carry out corresponding detection. Wherein, the whole reaction system is: the amount of the DNA template used was 5. Mu.L, and the total amount of the fluorescent PCR primer probe set and the hot-start Taq DNA polymerase premix was 15. Mu.L.
Specifically, in the fluorescent PCR primer probe group, the concentration of each of the two primers is 10 mu mol/L, and the dosage is 0.6 mu L; the concentration of the specific probe was 10. Mu. Mol/L and the amount was 0.4. Mu.L.
The hot start TaqDNA polymerase premix comprises 7.5. Mu.L of PCR buffer and 4.0. Mu.L of enzyme mixture, and ddH for the remainder of the make-up 2 O (1.9. Mu.L). Wherein, more specifically, the PCRBuffer comprises the following components: 0.25M Tris (pH 8.6), 50mmol/L KCl,24mmol/L MgCl 2 The method comprises the steps of carrying out a first treatment on the surface of the 10mmol/L dNTP,0.25mmol/L dUTP;7% glycerol, 2% formamide, 2.5% DMSO,0.05% NP-40,7. Mu.g/mL bovine thrombin, 0.5M proline, 10 ng/. Mu.L of single-chain binding protein (gp 32), 8mmol/L dithiothreitol. The enzyme mixed solution comprises the following components: 2.4U/. Mu.L of an RNase inhibitor; 0.5U/20. Mu.L uracil glycosylase (UDG enzyme); rapid Taq enzyme 0.15U/. Mu.L. The above-mentioned raw materials may be adjusted within a certain range, and the present invention is not limited to the specific amounts used herein.
Furthermore, in the actual amplification process of the invention, even if a one-step method (as described above, that is, the step-by-step addition of each component is not needed) is adopted to realize fluorescent PCR amplification, the full premix containing the components (comprising fluorescent PCR primer probe group and hot start Taq DNA polymerase premix) provided by the application still has a good amplification effect. Fully illustrates that the full premix liquid of the application has simple and convenient operation, good stability and no reduction of amplification sensitivity compared with the conventional step operation.
In the actual use process of the kit, the volume of the pre-mixed solution of the Taq DNA polymerase for hot start is three times that of the negative quality control product and the positive quality control product. For example, in a specific kit, the enzyme premix solution has a format of 825. Mu.L/tube X1, the negative quality control has a format of 275. Mu.L/tube X1, and the positive quality control has a format of 275. Mu.L/tube X1. Of course, the present invention is not limited to this ratio.
The invention also provides a quantitative detection method of haemophilus parasuis for non-disease diagnosis or treatment, which adopts the fluorescent PCR detection kit.
Preferably, the quantitative detection method includes:
s100, extracting DNA of a sample to be detected;
s200, carrying out fluorescent amplification on the DNA extracted in the step S100 by adopting the fluorescent PCR detection kit;
s300, judging a sample to be detected according to a fluorescence amplification result; and, in addition, the method comprises the steps of,
when the Ct value is less than or equal to 40, judging that the sample to be tested is positive to haemophilus parasuis;
when the Ct value is less than or equal to 45 and is 40, repeating the experiment once, and if the Ct value is still within the range or less than 40, judging that the sample to be detected is positive to haemophilus parasuis; if the Ct value of the repeated experiment is greater than 45, judging that the sample to be tested is negative to haemophilus parasuis;
and when the fluorescence amplification curve does not appear, judging that the sample is negative to haemophilus parasuis.
Here, the determination of the sample to be tested is based on the comparison of the detection results after qPCR detection in synchronization with the positive control and the blank control. In a more specific embodiment, the positive control is a positive control comprising Haemophilus parasuis (preferably comprisinginfBGene fragment) at a concentration of 10 5 Copy/. Mu.L; negative control was ddH 2 O。
The conditions of the fluorescent amplification reaction are: 2min at 37 ℃;10 s at 95 ℃;95℃for 1s and 60℃for 20s, 45 cycles. Fluorescence signals were collected starting from the 60℃step.
And judging the sample to be detected mainly according to the amplification curve and the reaction Ct value.
Preferably, the detection method further comprises: preparing a positive control plasmid; diluting the prepared positive control plasmid according to a gradient to obtain a standard sample; respectively carrying out fluorescent amplification on the obtained standard samples, then measuring Ct values, and constructing a concentration-Ct standard curve; wherein,
the step S300 further includes detecting a Ct value of the sample to be detected, and quantitatively detecting a concentration of the sample to be detected according to a concentration-Ct standard curve.
Embodiments of the present invention have the following advantages:
1. the primer probe group can specifically detect haemophilus parasuis of different serotypes, and the possibility of missed detection is reduced.
2. Compared with the traditional qPCR detection, the technical scheme of the invention has higher positive detection rate, and has remarkable improvement in detection time and pollution resistance. Provides reliable technical guarantee for rapidly and accurately realizing the detection of haemophilus parasuis.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIGS. 1 and 2 are graphs showing the amplification curve and melting curve of preparation example 1 of the present invention; wherein, FIG. 1 is an amplification curve graph, and FIG. 2 is a melting curve graph;
FIGS. 3 to 15 are amplification graphs of examples 4 to 7 of the present invention; wherein fig. 3 to 5 correspond to embodiment 4, fig. 6 to 8 correspond to embodiment 5, fig. 9 to 11 correspond to embodiment 6, and fig. 12 to 15 correspond to embodiment 7;
FIGS. 16 to 18 are amplification graphs of verification example 1 of the present invention; wherein, fig. 16, 17 and 18 are three sets of parallel experiments;
FIG. 19 is an amplification plot in verification example 2 of the present invention;
FIG. 20 is a concentration-Ct standard graph constructed according to verification example 2;
FIGS. 21 and 22 are amplification graphs of comparative example 1 of the present invention; wherein FIG. 21 corresponds to the amplification method of the present invention, and FIG. 22 corresponds to a conventional commercial kit;
FIGS. 23 and 24 are amplification graphs of the present invention verification example 3;
FIGS. 25 and 26 are amplification graphs of comparative example 2 of the present invention; wherein FIG. 25 corresponds to the amplification method of the present invention, and FIG. 26 corresponds to a conventional commercial kit;
FIGS. 27 to 31 are amplification graphs showing examples of application of the present invention.
Detailed Description
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The technical scheme of the invention is described in detail below with reference to specific embodiments. Wherein, the positive standard plasmid is obtained by adopting the following mode: by DNA recombination techniques (which can be carried out in a manner which will be understood by the person skilled in the art, for example, reference is made to the guidelines for molecular cloning experiments, author He Fuchu (translation), fourth edition]DNA recombination) and recombinant plasmid containing HPS specific gene fragment shown in SEQ ID No. 6. The concentration of the recombinant plasmid obtained was determined to be 166 ng/. Mu.L; according to the formula: y (copies/. Mu.l) = (6.02×10) 23 Copy number/mol) x (Xng/. Mu.L.times.10) -9 ) The copy number of the gene was calculated by/(DNA length. Times.660). And correspondingly diluting the recombinant plasmid according to the requirement to obtain the positive standard plasmid with the corresponding copy number.
In verification example 1 of the present invention, mention is made of and use of:
haemophilus Parasuis (HPS) is derived from haemophilus parasuis inactivated vaccine, and specifically adopts conventional 20 mL/bottle haemophilus parasuis disease tetravalent propolis inactivated vaccine (4 type SD02 strain +5 type HN02 strain +12 type GZ01 strain +13 type JX03 strain) produced by Shandong Hua Hong biological engineering Co., ltd, and the production batch number is 202209003;
pseudorabies virus (PRV) is derived from a porcine pseudorabies live vaccine, and specifically adopts a conventional porcine pseudorabies live vaccine (HB-98 strain) with the commercial specification of 10 parts per bottle produced by the Ministry of China and the industry, and the production batch number is 2212022;
porcine Parvovirus (PPV) is derived from porcine parvovirus inactivated vaccine, and is specifically a conventional porcine parvovirus inactivated vaccine (WH-1 strain) with the commercial specification of 20 mL/bottle produced by the WH family pre-organism stock company, and the production batch number is 20220817;
the porcine circovirus type 2 (PCV 2) is derived from porcine circovirus type 2 inactivated vaccine, specifically adopts porcine circovirus type 2 inactivated vaccine (SH strain) with the conventional commercial specification of 20 mL/bottle produced by Protect bioengineering Co., ltd, and has the production batch number of 072110006;
streptococcus Suis (SS) is derived from a streptococcus suis propolis inactivated vaccine, and specifically adopts a conventional and commercially available streptococcus suis propolis inactivated vaccine (BHZZ-L1 strain of streptococcus suis C group and BHZZ-L4 strain of streptococcus suis 2 type) with the specification of 20 mL/bottle produced by Shandong Hua Hong biological engineering Co Ltd, and the production batch number is 202209009;
the escherichia coli (ED) is derived from a piglet escherichia coli inactivated vaccine, and specifically adopts a piglet escherichia coli trivalent inactivated vaccine (containing K88, K99 and 987P cilia antigens) with the conventional commercial specification of 10 mL/bottle and produced by Shandong Hua Hong biological engineering Co., ltd, and the production batch number is 202209006;
the swine erysipelas bacillus (Ery) is derived from swine erysipelas live vaccine, the swine pasteurella multocida (PRDC) is selected from swine erysipelas live vaccine, and the swine erysipelas and swine pasteurellosis triple live vaccine (cell source +G4T10 strain +EO630 strain) with the conventional commercial specification of 10 parts per bottle produced by the middle-grazing industry Co., ltd are adopted, and the production batch number is 2208037, namely, in the actual test process, the swine erysipelas bacillus (Ery) and the swine pasteurella multocida (PRDC) are provided by the triple live vaccine;
actinobacillus pleuropneumoniae (APP) is derived from an inactivated vaccine for porcine infectious pleuropneumonia, and specifically adopts a conventional trivalent inactivated vaccine for porcine infectious pleuropneumonia (a serum type 1 9901 strain, a serum type 2 XT9904 strain, a serum type 7 GZ9903 strain) with the commercial specification of 20 mL/bottle, which is produced by the pre-organism stock company of the martial arts, and the production batch number is 20220706;
mycoplasma hyopneumoniae (Mp) is derived from an inactivated vaccine of Mycoplasma hyopneumoniae, and is specifically a conventional and commercially available inactivated vaccine (J strain) of Mycoplasma hyopneumoniae with the specification of 20 mL/bottle, which is produced by Shandong Hua Hong biological engineering Co., ltd, and the production batch number is 20220808;
porcine infectious Atrophic Rhinitis (AR) is derived from porcine atrophic rhinitis inactivated vaccine, and specifically adopts porcine atrophic rhinitis inactivated vaccine (Bo's bacillus JB) with conventional commercial specification of 20 mL/bottle produced by WU Han-family pre-organism stock Co., ltd 5 Strain), production lot number 20220602;
salmonella suis is derived from a piglet paratyphoid live vaccine, and specifically adopts a piglet paratyphoid live vaccine (salmonella C500 attenuated strain) with the conventional commercial specification of 10 parts per bottle produced by Shandong Hua Hong biological engineering Co., ltd, and the production batch number is 202208001;
the clostridium butyricum is specifically clostridium butyricum (type I) which is a conventional mixed feed additive with the commercial specification of 10 g/bottle and is produced by Zhengzhou Qifeng agriculture and technology Co., ltd, and the production date is 20230115;
enterococcus faecalis is specifically enterococcus faecalis (solid state I-003) which is a conventional mixed feed additive with the commercial specification of 500 g/bag and produced by Kunzea, biotechnology Co., ltd, and the production date is 20210801;
the bacillus subtilis is specifically used as a feed culture additive with the conventional commercial specification of 500 g/bag produced by the North sea industrial and vigorous biotechnology limited company, and the production date is 20221201.
In the invention, the real-time fluorescent quantitative PCR detection is carried out by adopting a real-time fluorescent quantitative PCR system-StepOnEPlus of the Siemens technology Co.
It should be further noted that, in the embodiment of the invention, all the PCR amplification is performed by directly using the full premix solution obtained by pre-mixing the primer probe set and the hot start Taq DNA polymerase premix solution, and the sequential mixing of the multiple components is not involved.
Preparation example 1, design and screening of primer probe set:
the first primer pair shown as SEQ ID No. 1 and SEQ ID No. 2, the second primer pair shown as SEQ ID No. 3 and SEQ ID No. 4, and the specific probe shown as SEQ ID No. 5 were specifically designed based on the HPS15 serotype gene columns published in GeneBank (Gene numbers: WCKA00000000, APCB01000037, NZ_APBU00000000, CP040243, CP021644, CP069308, CP049088, WCJR00000000, NZ_APBZ00000000, WIUS00000000, NZ_APBY00000000, CP005384, WCKJ00000000, WCJS00000000, NZ_APBX 0000; CP 009237).
SEQ ID No:1：5’-GCTTTCGATAATGCGACGTGCT-3’；
SEQ ID No:2：5’-TGCCGTTGAAAGCTCGTGTAAAGA-3’；
SEQ ID No:3：5’-GCCTGAAAGTCCTAATACTTCCACA-3’；
SEQ ID No:4：5’-GGCTTCAAGTAAGTCGTCAATCCCCATT-3’；
SEQ ID No:5：VIC- TACCACCTACGCCAGAGCCAACC -TAMRA。
The first primer pair and the second primer pair were initially screened using SYBR Green dye method [ 2X RealStar SYBR Mixture (with ROX), shenzhen Hui North Biotechnology Co., ltd.) and recommended reaction program thereof, and the Ct value and dissolution profile were analyzed to evaluate primer suitability and primer dimer condition. The results obtained are shown in FIGS. 1 and 2, wherein FIG. 1 is an amplification curve and FIG. 2 is a melting curve. Wherein F/R corresponds to the first primer pair and F2/R2 corresponds to the second primer pair.
As can be seen from the amplification curves and melting curves of FIGS. 1 and 2, the amplification of both sets of primers gave a typical S-type amplification curve, with no primer dimer and no non-specific amplification. Compared with the two primer pairs, the first primer pair has smaller detection Ct value and higher fluorescence value (shown in figures 1 and 2). Thus, in the subsequent examples, further operations were performed with the first primer pair in combination with a primer probe set constituted of a specific probe as shown in SEQ ID No. 5 as the subsequent preferred examples.
Preparation example 2, preparation of hot start Taq DNA polymerase premix:
the hot start Taq DNA polymerase premix comprises a PCR buffer and an enzyme mixture. The amount of the specific raw materials may be selected within a wide range, for example, specifically:
the PCR buffer includes: tris buffer with pH8.6 and concentration range of 200-500 mmol/L; the inorganic salt is chloride salt, and K can be selected + (i.e., potassium salt) and Mg 2+ (i.e., magnesium salt) at a concentration of 50mmol/L and 15-30mmol/L, respectively; dNTP of 0.25 mmol/L; dUTP of 0.25 mmol/L; 1-7% by volume of glycerol, 2-5% by volume of formamide, 2-4% by volume of DMSO, 0.05-0.5% by volume of NP-40,0.5-35 μg/mL of bovine thrombin, 0.2-8M of proline, 1-100 ng/. Mu.L of single-chain binding protein (gp 32), 1-10mmol/L of dithiothreitol.
The enzyme mixed solution comprises: the UDG enzyme is thermosensitive UDG enzyme with 0.1-0.5U; the addition amount of the RNase inhibitor is 2.4U/. Mu.L; the DNA polymerase is a hot start Taq DNA polymerase added in an amount of 3-6U, i.e., in a concentration of 0.15-0.3U/. Mu.L in a 20. Mu.L reaction system.
In more specific examples, the components and amounts of the PCR buffer of the present invention are shown in Table 1, and the enzyme mixture is shown in Table 2.
TABLE 1
TABLE 2
Example 1
The first primer set and the specific probe in preparation example 1 (wherein, in the whole 20. Mu.L reaction system, the concentration of each primer was 0.3. Mu.M, the concentration of the probe was 0.2. Mu.M, the annealing temperature was 60℃and the annealing time was 20 s) were used, and the hot-start Taq DNA polymerase premix in preparation example 2 had a copy number of 3.16X10, respectively 3 copies/μL、3.16×10 2 copies/μL、3.16×10 1 The copies/. Mu.L of positive standard plasmid was each subjected to fluorescent PCR amplification (the total reaction system in each fluorescent amplification reaction was 20. Mu.L, and the amount of positive standard plasmid used was 5. Mu.L). To the amplification efficiency,R 2 Correlation and detection reproducibility were examined, and the results obtained are shown in Table 3 under the number C.
Example 2
The procedure of example 1 was followed except that the hot start Taq DNA polymerase premix of example 1 was replaced with PCR Kit V1 manufactured by Hua Kui gold Biotechnology Co., ltd. For amplification efficiency, R 2 Correlation and detection reproducibility were examined and the results obtained are shown in Table 3 under the number A.
Example 3
The procedure of example 1 was followed, except that the following san Jose Biotech (Shanghai) Co., ltd was used: hieff Unicon Universal TaqMan multiplex qPCR master mix replaces the hot start Taq DNA polymerase premix in example 1. For amplification efficiency, R 2 Correlation and detection reproducibility were examined and the results obtained are shown in Table 3 under the number B.
TABLE 3 Table 3
As can be seen from Table 3, the primer pair probe set of the present invention can effectively identify a positive sample of Haemophilus parasuis. In addition, compared with the conventional commercial products, the hot-start Taq DNA polymerase premix phase can better adapt to the primer pair probe set of the invention, so that the whole qPCR-based detection system has the advantages of detection sensitivity, amplification efficiency and R 2 And detection reproducibility, are superior to those of a reaction system employing conventional commercial products.
Example 4
Respectively 3.16X10 3 copies/μL、3.16×10 2 copies/μL、3.16×10 1 The positive standard plasmid of copies/. Mu.L was used as a DNA template, the concentration of the probe was kept constant at 0.1. Mu.M throughout the system, and the concentration of each primer was adjusted to be 0.1. Mu.M, 0.2. Mu.M and 0.3. Mu.M, respectively, and fluorescent PCR amplification was performed in the same manner as in example 1. Each test under the same conditions was repeated three times in parallel. And detecting the amplification result to obtainThe results are shown in Table 4. The resulting amplification plots are shown in FIGS. 3-5, where FIG. 3 corresponds to a primer concentration of 0.1. Mu.M, FIG. 4 corresponds to a primer concentration of 0.2. Mu.M, and FIG. 5 corresponds to a primer concentration of 0.3. Mu.M.
Example 5
The procedure of example 4 was followed, except that the concentration of each primer in the whole system was kept constant at 0.3. Mu.M, the probe concentration was adjusted, and fluorescent PCR amplification was performed using probe concentrations of 0.1. Mu.M, 0.15. Mu.M and 0.2. Mu.M, respectively. Each test under the same conditions was repeated three times in parallel. And the amplification results were examined, and the results obtained are shown in Table 4. The resulting amplification plots are shown in FIGS. 6-8, where FIG. 6 corresponds to a probe concentration of 0.1. Mu.M, FIG. 7 corresponds to a probe concentration of 0.15. Mu.M, and FIG. 8 corresponds to a probe concentration of 0.2. Mu.M.
Example 6
The procedure of example 4 was followed, except that the concentration of each primer in the whole system was kept at 0.3. Mu.M, the concentration of the probe was kept at 0.2. Mu.M, the annealing time was kept at 20s, and the annealing temperatures were 58℃and 60℃and 62℃respectively, to carry out fluorescent PCR amplification. Each test under the same conditions was repeated three times in parallel. And the amplification results were examined, and the results obtained are shown in Table 4. The resulting amplification plots are shown in FIGS. 9-11, where FIG. 9 corresponds to an annealing temperature of 58℃and FIG. 10 corresponds to an annealing temperature of 60℃and FIG. 11 corresponds to an annealing temperature of 62 ℃.
Example 7
The procedure of example 6 was followed, except that the annealing temperature was kept at 60℃and the annealing times were 10s, 15s, 20s and 30s, respectively, to carry out fluorescent PCR amplification. Each test under the same conditions was repeated three times in parallel. And the amplification results were examined, and the results obtained are shown in Table 4. The resulting amplification plots are shown in FIGS. 12-15, where FIG. 12 corresponds to an annealing time of 10s, FIG. 13 corresponds to an annealing time of 15s, FIG. 14 corresponds to an annealing time of 20s, and FIG. 15 corresponds to an annealing time of 30 s.
TABLE 4 Table 4
As can be seen from FIGS. 2 and 4, the fluorescence detection signal intensity is lower at the primer concentration of 0.1. Mu.M and 0.2. Mu.M, and the fluorescence detection signal intensity is stronger and the detection Ct value and amplification efficiency are higher at the primer concentration of 0.3. Mu.M; at a probe concentration of 0.2. Mu.M, the amplification curve has a good linear relationship. The amplification reaction has a good linear relationship and the amplification efficiency is also in a qualified range when the degradation temperature is 60 ℃, and has a good repeatability, amplification efficiency and linear relationship when the annealing time is set to 20s. The final concentrations of primer and probe were thus determined to be 0.3. Mu.M and 0.2. Mu.M, respectively; the annealing temperature was 60℃and the annealing time was set at 20s.
Verification example 1
The 11 pathogens were included: haemophilus Parasuis (HPS), pseudorabies virus (PRV), porcine Parvovirus (PPV), porcine circovirus type 2 (PCV 2), streptococcus Suis (SS), escherichia coli (ED), erysipelothrix rhusiopathiae (Ery), actinobacillus pleuropneumoniae (APP), mycoplasma hyopneumoniae (Mp), infectious Atrophic Rhinitis (AR) of swine, salmonella and pasteurella suis (PRDC), 3 swine companion probiotics: clostridium butyricum, enterococcus faecalis and bacillus subtilis. Each of the amplification parameters [ i.e., the first primer pair and the specific probe in preparation example 1 (wherein the concentration of each primer was 0.3. Mu.M, the concentration of the probe was 0.2. Mu.M, the annealing temperature was 60℃and the annealing time was 20s ] according to the examples, and the hot-start Taq DNA polymerase premix in preparation example 2 ] was subjected to fluorescent PCR amplification. Each test under the same conditions was repeated three times in parallel. Wherein, the nucleic acid extraction of the pathogen and the probiotics is obtained by extracting a conventional commercial virus DNA/RNA nucleic acid extraction kit and a bacterial DNA extraction kit of the Siruisi biotechnology (Jilin) limited company. The results obtained are shown in FIGS. 16 to 18. As can be seen from the three groups of parallel test results in the figures 16-18, the kit provided by the invention has no amplification on 14 pig farm easy-to-see microorganism positive samples, and shows that the real-time fluorescence quantitative PCR detection kit for the HPS of the haemophilus parasuis has good specificity.
Verification example 2
Diluting positive standard plasmid with 10-fold gradient to obtain concentration of 3.16X10 -2 copies/μL-3.16×10 6 Nine gradients of positive standard plasmids of copies/. Mu.L were amplified according to the amplification parameters of example 1, and the results obtained are shown in FIG. 19, wherein the curves in FIG. 19 are sequentially from 1 to 9, and the corresponding concentrations thereof are sequentially decreased. As can be seen from FIG. 19, the present invention has a minimum limit of 3.16X10 of detection of the plasmid containing the HPS objective gene - 1 copies/μL（316 copies/mL）。
Further, the number of copies based on positive plasmid was 3.16X10 1 copies/μL-3.16×10 5 In the amplification result of copies/. Mu.L, the logarithmic value and the Ct value show good logarithmic linearity. Specifically, the slope of a standard curve of a real-time fluorescence quantitative PCR reaction system is-3.415, the intercept is 19.669, R 2 =0.999, amplification efficiency was 96.257%. On the basis, a standard concentration curve equation between the two is further constructed: y= -3.415x+19.669, where x is the log value of the concentration and y is the Ct value. As shown in particular in fig. 20. Based on this, quantitative analysis of HPS can be performed based on the Ct value measured.
Verification example 3, precision verification:
at 3.16X10 4 copies/μL-3.16×10 2 Each concentration gradient of the copies/mu L is detected for 3 times, the precision in the batch is a qPCR reaction system prepared by the same batch, each template is provided with 4 times of repetition, qPCR amplification is carried out, the precision between batches is that 5 batches of qPCR reaction systems are respectively prepared on 5 working days in a week, each template is provided with 2 times of repetition, and qPCR amplification is carried out; to determine the precision of the reagents of the invention. The formula for calculating the coefficient of variation is as follows: coefficient of Variation (CV) =standard deviation SD/mean×100%. The results are shown in Table 5, FIG. 23 and FIG. 24. Verified at 3.16X10 4 copies/μL-3.16×10 2 Within the range of copies/. Mu.L, the intra-batch CV of the present invention<0.5% and inter-batch CV<0.8%, and the precision is good. The results of the intra-batch precision test are shown in FIG. 23 and FIG. 24, respectively.
TABLE 5
Verification example 4
In order to evaluate the stability of the detection reagent, the present example used a thermal acceleration test, the prepared fully premixed working solution of the present invention was stored at 37℃for 5 days, and then used at a concentration of 3.16X10 2 qPCR amplification was performed on the copies/. Mu.L standard plasmid, and the test was continued, and the Ct value was recorded. The full premix reaction solution stored at-20 ℃ is used as a control group.
The results showed that the CT values measured in succession 1, 3, 5 were uniformly distributed between 28.5 and 29.0, with a coefficient of variation CV of less than 0.5% (Table 6). Therefore, the full-premix working solution of the research has higher stability and can meet the requirement of experiments.
TABLE 6
Application example
In order to accurately compare the detection effect of the reagent designed by the invention with that of the existing commercial reagent, the reagent of the invention is subjected to parallel comparison detection with the commercial reagent of Guangzhou Weibo Xin biotechnology Co. The method and the control method are used for detecting 124 parts of samples in parallel, wherein the samples to be detected comprise 48 parts of pig serum, 24 parts of nasopharyngeal swab, 52 parts of tissue samples such as meat, lymph and the like; in order to ensure the accuracy of the detection result, a positive control provided by the kit is set, a negative control for nucleic acid extraction is set to monitor the nucleic acid extraction process, an open-tube control is set to monitor the extraction environment, a PCR negative control is set, and the PCR reaction process is monitored.
HPS positive 23 cases were co-detected using the methods of the invention (FIGS. 27-28). Specifically, in the invention, 10 blood positives, 6 swab positives, 7 tissue samples such as meat and lymph are detected together, the positive rate of all samples is 18.5%, 8 blood positives, 6 swab positives, 7 tissue samples such as meat and lymph are detected together by the competitive product reagent, and the positive rate of all samples is 16.9%.
The positive samples detected by the invention are generally smaller than the Ct value of the same positive sample detected by a control kit by 2-3 (shown in figures 29-30), wherein the detection of the invention is weak positive (Ct values are respectively 34.84 and 33.84), and the detection results of the control reagent on the 2 weak positive samples detected by the invention are negative; for these 2 samples, detection was performed according to the method provided by the "SN/T4230-2015 Haemophilus parasuis disease quarantine technical Specification" (two replicates were set for each sample), and the detection result was positive, and was confirmed to be HPS positive (see FIG. 31).
The positive detection rate of the HPS of the detection reagent is 18.5%, the positive detection rate of the HPS of the competitive product reagent is 16.9%, and the results meet the popular characteristics of HPS in the current stage of China. Compared with competing products, the invention has higher positive detection rate, adopts fully premixed qPCR working solution, has simpler operation, and has shorter qPCR amplification time, thereby being very suitable for early, rapid and accurate detection of the HPS swine serious bacterial infection.
Comparative example 1
HPS genomic DNA was extracted and diluted 10-fold by 10-fold ratio 0 -10 6 Dilution of DNA as template (ddH 2 O is a negative control).
The DNA templates of the above-mentioned extracted and diluted gradients were detected by using a conventional commercial kit (qPCR reaction system of the reagent: 25. Mu.L, wherein the reaction solution of sterile nuclease-free water 2.0. Mu. L, PCR is 12.5. Mu.L, the enzyme mixture is 1.0. Mu.L, the fluorescent probe is 4.5. Mu.L and the DNA template is 5. Mu.L; the reaction conditions are 95℃2min;95℃15s,60℃30s,45 cycles), and the 10 HPS can be detected at the lowest 4 Nucleic acid dilutions, at 10 0 -10 4 The amplification efficiency of the standard curve in the gradient range is 93.595%, and the correlation coefficient R 2 >0.993. The amplification curve obtained is shown in FIG. 22.
Whereas amplification detection was performed according to the amplification parameters of example 1, at least 10 of HPS could be detected 6 Nucleic acid dilutions, at 10 0 -10 5 Gradient rangeThe amplification efficiency of the standard curve in the periphery is 98.395%, and the correlation coefficient R 2 >0.999. The amplification curve obtained is shown in FIG. 21.
Two parallel experiments were performed for each of the above operations. The results obtained are shown in Table 7.
TABLE 7
As can be seen from the above comparative examples, the detection method of the present invention has significant advantages in terms of detection sensitivity and amplification efficiency over conventional commercial products.
Comparative example 2
At 3.16X10 4 Mixing the copies/mu L positive standard plasmid with 100 mu L endogenous and exogenous interfering substances, simulating a sample, and performing qPCR amplification by taking the extracted nucleic acid as a template; each simulated sample was repeated 2 times.
The results are shown in Table 8, FIG. 25 and FIG. 26. The result shows that exogenous interferents such as antibiotics, antiviral drugs, microecologics and mucins have no influence on qPCR detection; in the case that a plurality of tissue samples (such as liver and lung) of endogenous interfering substances, milk samples and the like have no obvious influence on detection, and blood, excrement and feed have inhibition on the qPCR reaction of the invention, compared with a control group, the average value of the detection Ct is increased by 2-3. And the control kit of Guangzhou Weibo Xin biotechnology limited company has poor tolerance to ribavirin particles and amantadine hydrochloride mixed solution, feed, pig liver, lung, pig small intestine, blood, mucin and feces, and the average value of the detected Ct is increased by 3-4. The amplification results using the amplification method of the present invention are shown in FIG. 25, and the amplification results using the control kit of Guangzhou Wei Xin Biotechnology Co., ltd are shown in FIG. 26.
TABLE 8
In Table 6, T1 and T2 represent 2 experiments on one sample, respectively. One to five groups are exogenous interferents. The first group contains ceftiofur sodium for injection (300 mu L), florfenicol powder (30 mu L), tilmicosin premix (30 mu L) and normal saline (240 mu L), the second group contains amoxicillin for injection (300 mu L), doxycycline hydrochloride soluble powder (150 mu L) and tiamulin fumarate soluble powder (150 mu L), the third group contains dexamethasone acetate tablet (2.5 mu L), gentamicin sulfate (30 mu L), normal saline (567.5 mu L), the fourth group contains ribavirin particles (10 mu L), amantadine hydrochloride tablet (15 mu L) and normal saline (575 mu L), and the fifth group is feed residues. The sixth to thirteenth groups are endogenous interferents; the sixth group is a liver sample, the seventh group is a pig lung sample, the eighth group is a pig throat swab sample, the ninth group is a pig small intestine sample, the tenth group is a pig manure swab, the eleventh group is pig milk, the twelfth group is pig blood, and the thirteenth group is mucin (270 mu L) and physiological saline (270 mu L). Five to twelve groups of sample processing methods refer to: GB/T19915.7-2005.
Based on this, the invention has at least the following advantages:
1. according to the invention, primers and detection probes are designed according to the 15 serotypes of refB gene conservation regions of haemophilus parasuis, and the 15 serotypes of haemophilus parasuis can be specifically detected; detection of different bacterial serotypes that can be covered reduces the likelihood of missed detection.
2. As is well known, in a PCR reaction system, a primer, an enzyme mixture and a PCR buffer are generally stored separately, and when in use, the components are mixed in sequence and then used, so that the operation complexity is greatly improved by the operation mode; if all the components are mixed and then directly used in PCR amplification, the reaction system is unstable, and the detection result and the detection sensitivity are reduced. The invention adopts the independently developed full premix, thereby greatly saving the cost, and the full premix formula is obtained by optimizing the amplification target gene fragment and the specific primer probe sequence according to the invention, and has higher detection sensitivity. In the detection process, the template is only required to be added into the full premix in one step, so that the machine can be used for testing. And under the condition of ultra-fast qPCR reaction, qPCR amplification is completed within 30 minutes, which saves nearly half of the time compared with the traditional qPCR detection; while the detection sensitivity is as high as 316 copies/mL. The method can perform bacterial qualitative analysis and bacterial quantitative analysis at the same time, has good quantitative linear range (the slope of a standard curve of a specific real-time fluorescent quantitative PCR reaction system is-3.415, the intercept is 19.669, the R2=0.999 amplification efficiency is 96.257%), and has strong anti-interference capability, high detection precision and strong stability.
3. In the research, a UNG enzyme anti-pollution system is added in a qPCR amplification system, so that false positives of detection results caused by nucleic acid amplicon aerosol are avoided.
4. The detection of 124 cases of clinical samples shows that the method has higher positive detection rate than the traditional qPCR technology; compared with the prior art, the method has remarkable advantages in terms of operation steps, detection time and pollution resistance.
Therefore, the ultra-fast and high-precision qPCR technology established by the research provides a new method for fast and accurate detection of HPS, and provides a reliable technical guarantee for accurate detection of HPS. In addition, the invention provides the real-time fluorescence PCR detection primer pair, the probe, the kit and the detection method of the HPS, which not only shortens the detection time, but also has high detection precision and potential application value.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (10)
1. The fluorescent PCR primer probe group for quantitatively detecting haemophilus parasuis is characterized by comprising a group of primer pairs and a specific probe shown as SEQ ID No. 5; and, in addition, the method comprises the steps of,
the primer pair is selected from a first primer pair shown as SEQ ID No. 1 and SEQ ID No. 2 or a second primer pair shown as SEQ ID No. 3 and SEQ ID No. 4.
2. The fluorescent PCR primer probe set of claim 1, wherein the primer pair is selected from the group consisting of a first primer pair as set forth in SEQ ID No. 1 and SEQ ID No. 2.
3. The fluorescent PCR primer probe set of claim 1 or 2, wherein the 5' end of the specific probe is labeled with a fluorescent reporter group;
and/or, the 3' end of the specific probe is marked with a fluorescence quenching group;
the fluorescent reporter group is selected from one of 6-FAM, HEX, TET, cy3, cy5, cy5.5, texas Red, yakima Yellow and VIC;
and/or the fluorescence quenching group is selected from TAMRA, MGB, BHQ-1, BHQ-2 and BHQ-3.
4. The fluorescent PCR primer set of claim 3 wherein the fluorescent reporter group is VIC and the fluorescent quencher group is TAMRA.
5. A fluorescent PCR detection kit for quantitatively detecting haemophilus parasuis, comprising the fluorescent PCR primer probe set of any one of claims 1-4, and a hot start Taq DNA polymerase premix; and, in addition, the method comprises the steps of,
the hot start Taq DNA polymerase premix at least comprises a PCR buffer and an enzyme mixed solution;
in the fluorescent PCR detection kit, the fluorescent PCR primer probe group and the hot start Taq DNA polymerase premix exist in a full premix mode.
6. The fluorescent PCR detection kit according to claim 5, wherein the volume ratio of the PCR buffer to the enzyme mixture is 1.5-3:1;
and/or, the PCR buffer comprises: tris buffer with concentration of 200-500mmol/L, KCl with concentration of 40-60mmol/L and MgCl with concentration of 15-30mmol/L 2 dNTP with the concentration of 0.2-0.3mmol/L, dUTP with the concentration of 0.2-0.3mmol/L, glycerin with the concentration of 1-7 vol%, formamide with the concentration of 2-5 vol%, DMSO with the concentration of 2-4 vol%, NP-40 with the concentration of 0.05-0.5 vol%, bovine thrombin with the concentration of 0.5-35 mu g/mL, proline with the concentration of 0.2-8mol/L, single-chain binding protein with the concentration of 1-100 ng/mu L and dithiothreitol with the concentration of 1-10 mmol/L;
and/or, the enzyme mixed solution comprises: RNase inhibitor at a concentration of 2.2-2.6U/. Mu.L, uracil glycosylase at a concentration of 0.1-0.5U/. Mu.L, fast Taq enzyme at a concentration of 0.1-0.35U/. Mu.L.
7. The fluorescent PCR detection kit according to claim 5 or 6, wherein in the fluorescent PCR detection kit, the concentration of each primer in the primer pair is 0.20 to 0.40. Mu. Mol/L and the concentration of the specific probe is 0.15 to 0.25. Mu. Mol/L, respectively.
8. A method for quantitative detection of haemophilus parasuis for non-disease diagnosis or treatment, characterized in that the quantitative detection method employs the fluorescent PCR detection kit according to any one of claims 5-7.
9. The quantitative detection method according to claim 8, characterized in that the quantitative detection method comprises:
s100, extracting DNA of a sample to be detected;
s200, carrying out fluorescent amplification on the DNA extracted in the step S100 by adopting the fluorescent PCR detection kit;
s300, judging a sample to be detected according to a fluorescence amplification result; and, in addition, the method comprises the steps of,
when the Ct value is less than or equal to 40, judging that the sample to be tested is positive to haemophilus parasuis;
when the Ct value is less than or equal to 45 and is 40, repeating the experiment once, and if the Ct value is still within the range or less than 40, judging that the sample to be detected is positive to haemophilus parasuis; if the Ct value of the repeated experiment is greater than 45, judging that the sample to be tested is negative to haemophilus parasuis;
and when the fluorescence amplification curve does not appear, judging that the sample is negative to haemophilus parasuis.
10. The quantitative detection method according to claim 9, wherein the detection method further comprises: preparing a positive control plasmid; diluting the prepared positive control plasmid according to a gradient to obtain a standard sample; respectively carrying out fluorescent amplification on the obtained standard samples, then measuring Ct values, and constructing a concentration-Ct standard curve; wherein,
step S300 also includes detecting Ct value of the sample to be detected, and quantitatively detecting concentration of the sample to be detected according to the concentration-Ct standard curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410251918.7A CN117844956A (en) | 2024-03-06 | 2024-03-06 | Fluorescent PCR primer probe set, kit and method for quantitatively detecting haemophilus parasuis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410251918.7A CN117844956A (en) | 2024-03-06 | 2024-03-06 | Fluorescent PCR primer probe set, kit and method for quantitatively detecting haemophilus parasuis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117844956A true CN117844956A (en) | 2024-04-09 |
Family
ID=90536521
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410251918.7A Pending CN117844956A (en) | 2024-03-06 | 2024-03-06 | Fluorescent PCR primer probe set, kit and method for quantitatively detecting haemophilus parasuis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117844956A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174653A (en) * | 2011-03-01 | 2011-09-07 | 广西壮族自治区兽医研究所 | Haemophilus parasuis real-time fluorescent quantitative PCR (polymerase chain reaction) detection method |
| CN103205494A (en) * | 2013-03-29 | 2013-07-17 | 广西壮族自治区兽医研究所 | LAMP (loop-mediated isothermal amplification) method for detecting haemophilus parasuis |
| CN108977512A (en) * | 2018-08-03 | 2018-12-11 | 暨南大学 | Primer and probe and its kit and method based on digital pcr technology detection haemophilus parasuis |
| CN109593869A (en) * | 2019-01-26 | 2019-04-09 | 沈阳农业大学 | A kind of RPA primer for detecting haemophilus parasuis, RPA probe, kit and nucleic acid detection method |
| CN110408714A (en) * | 2019-08-30 | 2019-11-05 | 苏州西山生物技术有限公司 | Haemophilus parasuis body real-time fluorescence quantitative PCR kit and primer |
-
2024
- 2024-03-06 CN CN202410251918.7A patent/CN117844956A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174653A (en) * | 2011-03-01 | 2011-09-07 | 广西壮族自治区兽医研究所 | Haemophilus parasuis real-time fluorescent quantitative PCR (polymerase chain reaction) detection method |
| CN103205494A (en) * | 2013-03-29 | 2013-07-17 | 广西壮族自治区兽医研究所 | LAMP (loop-mediated isothermal amplification) method for detecting haemophilus parasuis |
| CN108977512A (en) * | 2018-08-03 | 2018-12-11 | 暨南大学 | Primer and probe and its kit and method based on digital pcr technology detection haemophilus parasuis |
| CN109593869A (en) * | 2019-01-26 | 2019-04-09 | 沈阳农业大学 | A kind of RPA primer for detecting haemophilus parasuis, RPA probe, kit and nucleic acid detection method |
| CN110408714A (en) * | 2019-08-30 | 2019-11-05 | 苏州西山生物技术有限公司 | Haemophilus parasuis body real-time fluorescence quantitative PCR kit and primer |
Non-Patent Citations (5)
| Title |
|---|
| 于江等: "副猪嗜血杆菌荧光定量PCR检测技术的建立与应用", 《家畜生态学报》, vol. 31, no. 4, 15 July 2010 (2010-07-15), pages 77 - 81 * |
| 刘爱平等: "《细胞生物学荧光技术原理和应用》", vol. 1, 28 February 2007, 中国科技大学出版社, pages: 282 - 285 * |
| 姜军平等: "实用PCR基因诊断技术", 31 August 1996, 兴界图书出版社西安公司, pages: 278 * |
| 梁乔等: "副猪嗜血杆菌PCR检测方法的建立及应用", 《现代畜牧兽医》, no. 9, 15 September 2018 (2018-09-15), pages 10 - 13 * |
| 罗宝正等: "TaqMan探针荧光 PCR检测副猪嗜血杆菌方法的建立和应用", 《江苏农业学报》, vol. 27, no. 6, 31 December 2011 (2011-12-31), pages 1 - 4 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111363858B (en) | 2019-nCoV S gene detection nucleic acid composition, kit and production method | |
| CN107937578A (en) | The primer combination of probe and kit of 15 kinds of respiratory tract infection pathogen joint inspections | |
| WO2023109032A1 (en) | Multiple nucleic acid detection system, and preparation method therefor and use thereof | |
| WO2022089550A1 (en) | Novel compositions and methods for coronavirus detection | |
| Liu et al. | Rapid and sensitive detection of Mycoplasma hyopneumoniae by recombinase polymerase amplification assay | |
| CN112359145B (en) | Multiple primers and kit for rapidly detecting influenza A, influenza B and novel coronavirus | |
| CN113005226A (en) | Oligonucleotide and kit for detecting SARS-CoV-2 | |
| CN104561340A (en) | Primers and kit for detecting aminoglycoside drug-resistance genes of bacteria | |
| CN113046452B (en) | Composition for detecting Boeck hollandia farci and application thereof | |
| CN114934125A (en) | Composition, kit and method for simultaneously detecting Brucella in sheep, cattle and pigs | |
| CN111363859B (en) | Nucleic acid composition for 2019-nCoV detection, kit and production method thereof | |
| CN110468223B (en) | Primer, probe, kit and method for detecting nucleic acid of mycoplasma pneumoniae and bauxiella based on dUTP/UNG method | |
| CN112941211A (en) | Multiplex fluorescence quantitative PCR detection kit for streptococcus suis type 2 virulence genes and detection method thereof | |
| CN117844956A (en) | Fluorescent PCR primer probe set, kit and method for quantitatively detecting haemophilus parasuis | |
| CN117844988B (en) | RT-qPCR detection primer and kit for porcine reproductive and respiratory syndrome virus and application of RT-qPCR detection primer and kit | |
| CN104561341B (en) | Primers and kit for detecting macrolide drug-resistance genes of bacteria | |
| CN109666750B (en) | Multiplex real-time fluorescence PCR detection primer probe set and detection method for identifying streptococcus suis and pasteurella multocida | |
| KR102209281B1 (en) | Simultaneous Pathogen Genes Detection Kit of Porcine Respiratory Complex Syndrome and diagnosis method of using this Detection Kit | |
| CN107385084A (en) | Application and its kit and detection method of the astragalus polyose in ring mediated isothermal amplification | |
| CN113416797A (en) | Fluorescent quantitative PCR detection kit for simultaneously detecting 7 types of adenoviruses | |
| Popoff | Detection of toxigenic clostridia | |
| CN108676900B (en) | Composite PCR typing kit for distinguishing eight porcine actinobacillus pleuropneumoniae serotypes and application thereof | |
| Yuzi et al. | Establishment and application of a real-time, duplex PCR method for simultaneous detection of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis | |
| CN113151591A (en) | Nucleic acid composition and kit for detecting SARS-CoV-2 | |
| CN110157836B (en) | Primer, probe and method for detecting IBRV and BVDV |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |