Single-tube multiple PCR (Polymerase Chain Reaction) pet pathogen detection kit

By using multiple PCR technology and the design of specific amplification primers in pets, combined with the optimization of the reaction system, efficient and specific detection of multiple pet pathogens under low NGS sequencing data is achieved, and the existing technology is solved The challenges of multiple amplification and high-throughput sequencing improve the sensitivity and specificity of the diagnosis.

CN119193882APending Publication Date: 2024-12-27SHANGHAI CISTRON BIOTECHNOLOGY CO LTD
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Patent Information

Application Number
CN202411511351.9
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2024-10-28
Publication Date
2024-12-27

AI Technical Summary

Technical Problem

The prior art is difficult to detect multiple pathogens efficiently and specifically in pets at the same time, especially during latency infections, and multiple amplification and high-throughput sequencing are required, and there are challenges in target region selection, primer design and reaction system optimization. .

Method used

Provide a single tube multiple PCR pet pathogen detection kit, which contains multiple pairs of specific amplification primers, and efficiently detect pathogenic microorganisms at a lower NGS sequencing data volume, combining specific primer designs , the composition of the reaction system and the buffer ion concentration optimization, so as to achieve specific amplification and detection of various pathogens.

Benefits of technology

It realizes efficient and specific detection of multiple pet pathogens at lower NGS sequencing data, improves the sensitivity and specificity of diagnosis, and can detect multiple pathogens at the same time, which is suitable for early pathogen screening of pets.

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Abstract

The invention discloses a multiple high-throughput sequencing detection kit for pet pathogenic microorganisms, and belongs to the technical field of microorganism detection. Important pathogens in pet samples are specifically detected, and samples such as oral swabs, anus swabs and blood of pets are specifically amplified and sequenced, so that the pet pathogens are rapidly screened.
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Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to a multiplex high-throughput sequencing detection kit for pet pathogenic microorganisms. Background Art

[0002] Although the isolation and identification of pathogens is the gold standard for diagnosis, some pathogens are zoonotic pathogens and pose safety risks. Therefore, it is difficult to implement them in pet clinics and hospitals. With the advancement of molecular biological diagnostic technology, related detection technologies are constantly optimized, and new detection technologies with simple operation, high sensitivity and strong specificity have emerged, such as real-time fluorescence quantitative PCR technology, isothermal amplification technology and gene chip and other nucleic acid detection technologies, colloidal gold immunochromatography technology, time-resolved fluorescence immunochromatography technology, fluorescent microsphere immunological detection technology, chemiluminescent immunoassay technology and immunobiosensor and other immunological detection technologies based on nucleic acid detection systems and other new detection technologies, which can meet the needs of rapid detection of pathogens in a variety of environments.

[0003] The simultaneous detection technology of multiple pathogens is also a development trend in clinical testing. Especially for animals in the incubation period of infection, pathogen screening is required in the early stages of the disease. This requires the multiple amplification of multiple pathogens, which is still a challenge for pet pathogens.

[0004] Pets go to the hospital for medical treatment mainly due to digestive system abnormalities such as vomiting and diarrhea, and respiratory system abnormalities such as coughing, runny nose, and sneezing. The above two types of abnormalities are mostly caused by pathogen infection. Among them, digestive system pathogens are mostly caused by plague virus, coronavirus, Toxoplasma, Cryptosporidium, etc., while respiratory system pathogens are mostly caused by adenovirus, herpes virus, calicivirus, mycoplasma, bronchiseptica, etc.

[0005] Through multiple PCR amplification, NGS detection of pathogens has been widely studied in human pathogen detection. By adding multiple groups of PCR products into a PCR reaction system and adjusting the reaction system, multiple target genes can be amplified simultaneously in one reaction system. In the subsequent steps, the amplified products are sequenced in large scale parallel, so that the amplified products can be detected by bioinformatics methods, thereby evaluating the pathogens contained in the sample and their relative abundance.

[0006] Since multiplex PCR requires amplification of multiple sites in one system, it is necessary to optimize the selection of target regions, primer design, reaction system, and reaction temperature. Summary of the invention

[0007] The present invention provides a kit for high-throughput detection of microbial nucleic acids based on multiplex PCR technology and high-throughput sequencing technology. The kit mainly comprises multiple pairs of specific amplification primers. Through multiplex PCR amplification of primers, pathogenic microorganisms can be detected efficiently and specifically with a relatively low amount of NGS sequencing data, thereby assisting in the diagnosis of microbial infection.

[0008] The invention provides a single-tube multiplex PCR pet pathogen detection kit, which can simultaneously detect Lyme disease, Helicobacter pylori, Bordetella, Chlamydia, Mycoplasma, Ehrlichia, Anaplasma, Mycoplasma, Giardia, Toxoplasma, Heartworm, Feline Herpesvirus Type I, Canine Adenovirus Type I, Canine Adenovirus Type II, Feline Parvovirus, Canine Parvovirus, Canine A Coronavirus Type I, Rabies Virus, Feline Immunodeficiency Virus, Feline Leukemia Virus, Feline Calicivirus, Feline A Coronavirus Type I, Haemophilus Mycoplasma, Canine Babesia, Bartonella henselae, Rickettsia, Bartonella clarridgeae, Rickettsia, Leptospira, Feline Cryptosporidium, Eperythrozoon, Canine Parainfluenza Virus, Canine Cryptosporidium, Tritrichomonas fetus, and Canine Distemper Virus.

[0009] In the implementation case, the primer length is 20-30 bp and the tm value is 55-65°C.

[0010] In the implementation case, the amplified region is a specific conserved region of the corresponding species.

[0011] In an embodiment, the primer pairs are used to amplify the corresponding species.

[0012] Primers 1 to 10, for Lyme disease detection; Primers 11 to 20, for Helicobacter pylori detection; Primers 21 to 30 were for Bordetella ; Primers 31 to 40 for Chlamydia; Primers 41 to 50 are for Ehrlichia ; Primers 51 to 60 are Anaplasma ; Primers 61 to 70 are for mycoplasma; Primers 71 to 80 were for Giardia lamblia; Primers 81 to 90 are for Toxoplasma gondii; Primers 91 to 100 are for heartworm; Primers 101 to 110 are feline herpesvirus type I; Primers 111 to 120 are canine adenovirus type I; Primers 121 to 130 are canine adenovirus type II; Primers 131 to 140 are for feline parvovirus; Primers 141 to 150 are for canine parvovirus; Primers 151 to 158 are canine coronavirus type I; Primers 159 to 168 are for rabies virus; Primers 169 to 178 are for feline immunodeficiency virus; Primers 179 to 188 are for feline leukemia virus; Primers 189 to 196 are for feline calicivirus; Primers 197 to 206 are feline coronavirus type I; Primers 207 to 216 are for Haemophilus influenzae; Primers 217 to 226 were for Babesia canis; Primers 227-236 are Bartonella henselae Primers 237 to 246 are for Rickettsia; Primers 247 to 256 for Bartonella clarridgeae ; Primers 257 to 266 are for Rickettsia; Primers 267 to 276 were for Leptospira; Primers 277 to 286 are for Cryptosporidium felis; Primers 287 to 296 were for Eperythrozoon; Primers 297 to 302 are canine parainfluenza virus; Primers 303 to 312 are for Cryptosporidium canis; Primers 313 to 322 were for Tritrichomonas foetus; Primers 323 to 332 are canine distemper virus; Primers 333~342 were internal reference genes.

[0013] In an embodiment, the primer set specifically amplifies multiple target regions of one or more species.

[0014] In an embodiment, the primers perform multiplex PCR amplification.

[0015] In multiplex PCR amplification, there is a positive correlation between primer concentration and amplification length. High concentrations of primers will affect each other. Therefore, the final concentration of primers is 0.05~0.4μM.

[0016] In the reaction system, since dNTPs can bind to magnesium ions, in order to ensure the reaction yield and sufficient free magnesium ions to catalyze DNA polymerase, the concentrations of magnesium ions and dNTPs need to be balanced, with 100~200μM dNTPs and 0.5~2mM magnesium chloride as the final concentrations.

[0017] In addition, since the product length is inversely proportional to the amplification salt ion concentration in terms of amplification efficiency, the potassium ion concentration is increased to 60~100mM for the amplification system of this length.

[0018] At the same time, since the amount of DNA polymerase affects the amplification yield and amplification balance, DNA taq enzyme was used for amplification, and its final reaction concentration was 0.2~2U.

[0019] Potassium ions are used to bind to the phosphate groups of the DNA backbone and stabilize the double-stranded formation. Ammonium ions can interact with the hydrogen bonds between bases and destroy the double-stranded bonds. Therefore, potassium chloride and ammonium sulfate are used as buffer ions in the PCR reaction system and to improve the specificity of primers.

[0020] In addition, some auxiliary reagents are usually selected to open the secondary structure, improve the amplification specificity, or protect the enzyme activity at high temperatures, such as non-ionic detergents (Tween 20, Triton X-100), bovine serum albumin (BSA), polyethylene glycol (PEG), dimethyl sulfoxide (DMSO), betaine, and glycerol for protecting low-temperature enzyme activity.

[0021] Therefore, in the final reaction system, the reaction components are as follows: 10~30 mM Tris-HCl, 60~100 mM KCl, 10~30mM (NH4)2SO4, 0.5~2.0 mM MgCl2, 0.1~0.2 mM dNTPs, 5~10% glycerol, 0.05~0.1% Tween® 20 5~10% polyethylene glycol (PEG), 1~5% dimethyl sulfoxide (DMSO), 0.2~2U Taq DNA Polymerase, pH 8.5~9.0.

[0022] In the implementation case, the primer amplification product is 150-250bp.

[0023] In the implementation case, the amplified product can be used to construct a library by methods such as end repair and A addition, sticky end ligation, etc.

[0024] In the implementation case, the length of the library including the adapter is about 270-370 bp.

[0025] The primer sequences are: SEQ ID 001 AAATCGTCGACATGAATATGCG SEQ ID 002 TGGGATGACTAAAGAAGTGGC SEQ ID 003 GCACCCCCATCTTTATCCCC SEQ ID 004 AGGCAGGGAAGTTGTTTGGG SEQ ID 005 CGATCCCAAATCTAAACAGTTAAGCC SEQ ID 006 TTCAAGAGGATTCCCTTCGGC SEQ ID 007 TGCTCTAGTGCTCTAGCTCAA SEQ ID 008 ACCAGTCCTATTGCACTCCA SEQ ID 009 TGATTTGGTGCTGGGAATATGGA SEQ ID 010 TCGGTGGTCAAGAATTCAGGA SEQ ID 011 AACACAAGATTAGCGCTTATGGG SEQ ID 012 GGCTCAAATCTTCTCCTGCG SEQ ID 013 AGCACACAAGAGAGTATTGGC SEQ ID 014 TAGTAATGGCGATCACCCC SEQ ID 015 AGGCTTTGTTTCCATCAAATACGG SEQ ID 016 ATGGAGTCGCTTTTAACGCC SEQ ID 017 TCTTCGTTAGAGTCTAAAAGCACGC SEQ ID 018 CTAACAGCGCGAGTGAATGCC SEQ ID 019 ACGATTTGGGCTTTGATGCG SEQ ID 020 TGAGCGGCTCTACAAATTCCC SEQ ID 021 AATTGGCCCTGTGGTTCCC SEQ ID 022 ACCTTCCAGGAAGCTGTGGG SEQ ID 023 TCGACGTCGGATGTTTTGCC SEQ ID 024 TATGCTCTTCGATCTCGCGC SEQ ID 025 TTGTGCTGTATCGAGGGAGCC SEQ ID 026 GCAGGTCATGATGGTCTCCG SEQ ID 027 CATTGAATTGCCAGGTGTCGG SEQ ID 028 ATCCGCTACCTGGTCTACCG SEQ ID 029 TGGCATCAAAATTCGTGCGG SEQ ID 030 GTTCGGAATAAGACGCTGCC SEQ ID 031 GGATGGAGCCTATGTCTTGCC SEQ ID 032 TGTTTCTCTACCAACAAGTTCGC SEQ ID 033 GTGCCGTGGATAAAGTTCCC SEQ ID 034 AGTTACAGCTCTCACTCACCC SEQ ID 035 AGATATACGGACGTAGCGCC SEQ ID 036 AAATGGGCTGGAGGTATCGG SEQ ID 037 TGGCATAACTCGATTTAAAGGC SEQ ID 038 CACGTATCTTGACGAGATCGC SEQ ID 039 TGTTCGCCAGCAATATTCCC SEQ ID 040 GTGCATCATCGAGGAAAAAGGG SEQ ID 041 TAGTTGGCATTAATTCAACAGGGG SEQ ID 042 CCAATTATCCTTGCTATCATCTTTCCG SEQ ID 043 GTCAAAGAGGAAAAGCAGAGGG SEQ ID 044 GTCCAGAACTTACATCATGTAAACCC SEQ ID 045 AGAGCAACACCAATATCTAAACACA SEQ ID 046 TGAATTGCAGTTTGTGAAGTAATGT SEQ ID 047 AGGTAGGTCGTATGTCTAAAGTGA SEQ ID 048 ATTCGCCGGTAGTTCCACAT SEQ ID 049 TCCATTCCTGAGAAGTAATAACCACA SEQ ID 050 AGTTTCAGCAGAACAATTGGGAA SEQ ID 051 TTGGTGGTAACTTCGTGGGC SEQ ID 052 GGTACCCGTATTGAGCTGCC SEQ ID 053 TCCTTACGGTGATGTGTGGC SEQ ID 054 AACCCTGCTCATAACAAAGGC SEQ ID 055 CTACCAATCTTTCCAACTGAAGGG SEQ ID 056 ACTAAGAAGACCGTACCTCGC SEQ ID 057 GGGTATAAGGTGCCAGAGGG SEQ ID 058 CGGGCCATAGCATCTATAGCC SEQ ID 059 ACAAGAGCCGCCAATATCCC SEQ ID 060 ATGGCTTCAGCTGGTTTGCC SEQ ID 061 TCAGCCAAGATTTAGGTGACTGA SEQ ID 062 GCTCTACGGAACATTCAACCC SEQ ID 063 TCATCGCTGATAAAGGTATGAGCA SEQ ID 064 TTGTTTTCTGAAATGACCGTTTGG SEQ ID 065 TCACCTTTAAATGCACTAGCCTG SEQ ID 066 AACAAGCAGAAGTTTTCAATGGT SEQ ID 067 TGTTACACCATGGTCTGTGTGA SEQ ID 068 CCTGCTCCAATAGATGTAGAAGGA SEQ ID 069 CCTGCTCCAATAGATGTAGAAGGA SEQ ID 070 TGTTACACCATGGTCTGTGTGA SEQ ID 071 AAGTGATGGCTGTCAGTGGG SEQ ID 072 AGCGGTCCTGTATCTGTGGG SEQ ID 073 CCACGATCTCGAAGATGTGGG SEQ ID 074 TTGCAGAATCTGTCCCAGGG SEQ ID 075 CTTTGGAACTGTGCTGAGCG SEQ ID 076 CCTCTGCAGTAAGGTAGCGG SEQ ID 077 ACTTGAGTCTGTCTTGTCGGC SEQ ID 078 TGTATCTGATCGCGTCTGCC SEQ ID 079 GGTTGAGTAAATTGCCCGGG SEQ ID 080 AGGGTGCCCTAAATGTTGCC SEQ ID 081 CAAAGTTGCTTCACCATTGCC SEQ ID 083 TACGATAAGAGGCCTGACCC SEQ ID 084 AAGCGTCGAATAACTGTGGC SEQ ID 085 TTTTCAGTGGTTTGAAggcg SEQ ID 086 ATGTCTGTAGGTTTCGTCCG SEQ ID 087 TGAGCCACTTTGTAGCACCG SEQ ID 088 TACTTGTTCCGCATTCCGCC SEQ ID 089 AATGAGCAATGAACGTCGCG SEQ ID 090 GAGTGACTGAGATAGCTGGGG SEQ ID 091 aagtttcgAAGCAAAGTTTGGC SEQ ID 092 TGGTTGCACAACACTTTTGGC SEQ ID 093 GACTTGTTTCTTTGGTTCCGC SEQ ID 094 GCACATGCGAATGAATGGG SEQ ID 095 CAAACCATTCTAACTAACcggc SEQ ID 096 CACTGAAATCCACGGTTTCCC SEQ ID 097 TCATTCCTTCTCTGTGCcgat SEQ ID 098 TGGCAAGCATAACAGATAACAACT SEQ ID 099 CATCTGTCGTATCTCGCGCT SEQ ID 100 AGCAGCATCTAAAAGGCAGGA SEQ ID 101 GGTTTGATTATGATGGCCCCG SEQ ID 102 ATCACGAGCTCCATCAAGGC SEQ ID 103 CACCGTCTACGAAATCAGGC SEQ ID 104 TGTACCCAAACTGTGACGGG SEQ ID 105 CGGTCTCAAAACCAACTAACCC SEQ ID 106 GCAGACAAATACGCTATCTACCG SEQ ID 107 GAAGACCATTAGCCACCCCG SEQ ID 108 TTCGCATGTTCGAGAAAGCC SEQ ID 109 CCTATTGGGTCTTGGTAGGGC SEQ ID 110 TACTGCTTCATCTACCGCGG SEQ ID 111 TGCTTTTACCACTGTTTGCGG SEQ ID 112 AATTAACCCAAAGGCGTGGC SEQ ID 113 CGGTGGTTCTGGTATCAGGG SEQ ID 114 CAATTGATGCTGACCCACCG SEQ ID 115 CCCTATGTCATCCTGCTGCC SEQ ID 116 GGACATAGAAACGCAGGACCC SEQ ID 117 TGTCTTCCCAAGTGAGAGGC SEQ ID 118 ACCTGTGGTGTCTCTCTTCCC SEQ ID 119 AAAGAGCCGCAGAACTCACA SEQ ID 120 CTGCATGCTTAAACAGGCCC SEQ ID 121 ATTCTGGATATGCTAGCGCCC SEQ ID 122 TTGGAAGAGAAGGGGATGCG SEQ ID 123 ATGGGAGGTTGTCTCAGCG SEQ ID 124 CGCAAGCAATTTCTGAGGC SEQ ID 125 GGGTCAAGAGGTAGTGTCGG SEQ ID 126 TAAGGACACGTACCCATGCC SEQ ID 127 TGGCACGTGTTCATCAACCC SEQ ID 128 ACCTCTTGTCAAAGGTGCCC SEQ ID 129 AGGATGTGTTGGATCAATCTGGG SEQ ID 130 GCTTTGAATCTCGTGGAGCC SEQ ID 131 AGAGCCAGGAGGTAAGGG SEQ ID 132 GTCGTGTTCTTTTGCAGCGG SEQ ID 133 GGAGCAGTTCAACCAGACGG SEQ ID 134 GAGTTTGCTGTGATTTCCACCC SEQ ID 135 ATCCCATACTGGAACTAGTGGC SEQ ID 136 GCCCAATGCTCTATTTGTTTGCC SEQ ID 137 GTGGTTGGTTGGTTTGCCC SEQ ID 138 ATGTTGTGCATCTCCCCACCC SEQ ID 139 ACTCAGATTTTTGGTGGAAAGGT SEQ ID 140 TTCTAGGTGCTAGTTGAGATTTTCA SEQ ID 141 CAGTATACTGAGGAAGTTATGGAGGG SEQ ID 142 GGCGAGACTATCAACTTCCG SEQ ID 143 CGTGGTCCGAAATAGAGGCA SEQ ID 144 ACCTGGAGGCACAAGTCCTA SEQ ID 145 TGGAAAACGGATGGGTGGAA SEQ ID 146 CCCAAGCATTTGCATCAACCA SEQ ID 147 TTGAGGCGTCTACACAAGGG SEQ ID 148 CAGGTGTTTCTCCTGTTGTGG SEQ ID 149 TGTTGTTATGGTGTGGGTGGT SEQ ID 150 AAATTAGGGCGCAACGATTGA SEQ ID 151 TGTAGTTGTTGGTGGTTATTACCC SEQ ID 152 GGTTTACCTCGTGCATTGCC SEQ ID 153 TGCTACCAACGTATTTGCCCC SEQ ID 154 CTGCGACACCAAAACTGGG SEQ ID 155 GTGGCTGTGGCGAAAAAGAA SEQ ID 156 TGTGACGTGGGAGTCTGTTT SEQ ID 157 TCCATGTTCTTGGCCTGGTT SEQ ID 158 GTGTTGCACCAACCACTCTAAG SEQ ID 159 CAATCCTGTTGTCCAAGACCC SEQ ID 160 ACTGCCTTCTTATCGTTCGGG SEQ ID 161 GTCAGATCCCTAAATGCAACGG SEQ ID 162 TCTTTGTCAGTTCAGCCGCC SEQ ID 163 ATGTCGCGGTCAACTTTCCC SEQ ID 164 ATCCTCTTCATTGGTGGCCG SEQ ID 165 AGTCTTCAACGTTTCCAAACCC SEQ ID 166 ACAGGTCGTGAAGTCTTGCG SEQ ID 167 GTCAGATCCCTAAATGCAACGG SEQ ID 168 TCTTTGTCAGTTCAGCCGCC SEQ ID 169 TTAAAGCGCTAGCAGCTGCC SEQ ID 170 TATCTGTGGGAGCCTCAAGGG SEQ ID 171 GAAGATAAATTACAGGAAGAACCCCC SEQ ID 172 CTCAAGTCTGGAATAGCTTGGC SEQ ID 173 CCTGTCAGGACTTTCTTGGGG SEQ ID 174 CATTCCATCTTTTCCATATTCTGCC SEQ ID 175 TTCACCATATCTTAAGGCCCC SEQ ID 176 TTTCTGCCTTCTGATTACTGCC SEQ ID 177 GAAGGGAATTTCAGATGGGCC SEQ ID 178 TCATATTTAAAAGTCCTGCTACCGC SEQ ID 179 CCACCCTATTTGATGAGGCCC SEQ ID 180 CTCGGTACCCCTTATTGCCC SEQ ID 181 GCCAAAGAGAGGTACCTGGG SEQ ID 182 GCAGCTCTCTGTTACTTGCC SEQ ID 183 GCCAAAGAGAGGTACCTGGG SEQ ID 184 GCAGCTCTCTGTTACTTGCC SEQ ID 185 GGGAACACATTGTGGAGGGG SEQ ID 186 CCCAAGAGGCTTGTCTTCCC SEQ ID 187 GCCAAAGAGAGGTACCTGGG SEQ ID 188 GCAGCTCTCTGTTACTTGCC SEQ ID 189 AAGCGAAGGATGATTTGGGG SEQ ID 190 CAGAGCCTCAACACTAGGGC SEQ ID 191 GAATGGAGGGAACACAATGCC SEQ ID 192 CATAGCCCCGATCAACTGCC SEQ ID 193 GGATGAGACCTCCTTCTCGG SEQ ID 194 AGACCATGGCATTGTCTGGG SEQ ID 195 GAATGGAGGGAACACAATGCC SEQ ID 196 CATAGCCCCGATCAACTGCC SEQ ID 197 TTTGCACAGTTAATGCTTTGCC SEQ ID 198 CCTCAGGTTGGAGTGTACCC SEQ ID 199 ACAAAGTGGTGACTAGCGGC SEQ ID 200 ACCCCCTATAAGAGAGGCGG SEQ ID 201 TGGACACTGTCAAGTCTATCGG SEQ ID 202 GGCATAAGCTTTTTGTTCAGCC SEQ ID 203 GCTAGAGCTCGTACAGTGGG SEQ ID 204 TCCCATCCCATCAAACAGCC SEQ ID 205 TACAGGACCTGTGATAGCGG SEQ ID 206 CAAACCAGTATAGCAGATGTAGCC SEQ ID 207 CCACTTCCACTTCTAGTACCGG SEQ ID 208 TCCTTACTATCCGGATCATAAGCC SEQ ID 209 GGTCTACGGACTCCAAAGGG SEQ ID 210 TGTCAAACTTACCTCCTGCG SEQ ID 211 CAACAAAGGAAGAGAAGGCGG SEQ ID 212 TGAAAGCTGGAGATTTCTTTCCC SEQ ID 213 TAGTTCAAGCACTAGCACCCC SEQ ID 214 TGGAGGAGCTAAGCTTAGGG SEQ ID 215 GAGACCGAGGAGATAGTTGGG SEQ ID 216 GGATTCCGATGAAGAGGGGG SEQ ID 217 ACACCTTCGGATTCTTCTTCGG SEQ ID 218 TAGTGACCCTTGAAGTGTGCC SEQ ID 219 CACCGGTGAACTGAAAAGCG SEQ ID 220 GATGTAGCTGTTCCCTGACGG SEQ ID 221 ACTTCAGATATCGAGACCCGC SEQ ID 222 ATCCCTCCACACTGTAACCG SEQ ID 223 GAGTTGGACAGACAGCAGGC SEQ ID 224 ATCGGCAGGGACATACTCCG SEQ ID 225 CTGGATCATTATTGGCACTCGG SEQ ID 226 CGTGTACACTGCTTGATGGC SEQ ID 227 CCCGTTGATACTCTTCCCCC SEQ ID 228 TTGGGGGAGAGTATCAGCGG SEQ ID 229 TCTCAGATAATTGTGAGGGCG SEQ ID 230 GTGCATTCCCAACAAAATGGC SEQ ID 231 ACTACACTTCCTTTTACGCGC SEQ ID 232 TCATTGCAAACATTGTATCGCG SEQ ID 233 AAAGCCTTTGAAACGATGGC SEQ ID 234 ATTGTATTGATCGAGGAGGGC SEQ ID 235 GTTGGCAGTGTTGTTATTGGC SEQ ID 236 AACTGACCTCCATTGACTGCC SEQ ID 237 GTCAAATTGACAAGGAGCCG SEQ ID 238 CCCTTACCCACATGAATAGCC SEQ ID 239 TCAGCAACCTGTACTTGCCC SEQ ID 240 TTGAAGAAAAAGGTGTAGCTTTCGG SEQ ID 241 AAAAGCGAGGATAGTTGATTCGG SEQ ID 242 TCCTAACGTCAGTGCTACGC SEQ ID 243 TGAGACTAGGCACGAAGAGC SEQ ID 244 AGCGAACTGATTTAGTGCTAATTTCC SEQ ID 245 TGCCATCATCAAGAGCTAGAGG SEQ ID 246 CAAGAACAGCTTGCACCATT SEQ ID 247 TATGTTGAGAAACGGAGCCG SEQ ID 248 TTTTGGTCGTCCTCTTCCCC SEQ ID 249 TGCAGAACATATCCGTACGCC SEQ ID 250 TTTTGTTTCACACCTGCGGG SEQ ID 251 TCGTATCATATACAGCAAGCACAT SEQ ID 252 TGGCTTTTTGTATAAGGGCCA SEQ ID 253 AAGAGACCTCCGTCCTCCTC SEQ ID 254 TCGGTGTTTATGATTCACTCGAGA SEQ ID 255 CATCTTCGCTGTTTGGTACGT SEQ ID 256 TCTTGGCTAAAGAGGGTTTTGAT SEQ ID 257 CAGGATATGGATAAAGGATGGATGCC SEQ ID 258 GCAAATGCAGGGAGTGACGC SEQ ID 259 CGACGACTCGAATGTTTAACGC SEQ ID 260 AGTGATTCCAGCTGTGTCGG SEQ ID 261 GGAGACGCTACATTTGATTGGG SEQ ID 262 TCAAAACATCTCTGCAAGGCC SEQ ID 263 AGATGAAAATCGAGTCAGAGCG SEQ ID 264 TCTAATCTTAAGGATAGATAGGCCG SEQ ID 265 CAAAAAGTAAGCCGTGCCGT SEQ ID 266 ACGCAATTGAAGCACTCAGC SEQ ID 267 gagaaaagtgTAGTCTCCAAGCCG SEQ ID 268 GGAGTCGATGAAGAAGGTTCTCCG SEQ ID 269 GtcacaaaaagaaaatggcAGGG SEQ ID 270 cacACGCACCCTAATAAAGCC SEQ ID 271 CTTTGAGTGCTTTCGCTTCGG SEQ ID 272 CCGGAGCTCTTCTTCAAGGG SEQ ID 273 TGAATACCGCATACCAGTCCG SEQ ID 274 TCTCTTTATTCGGAGTAAGGTCGG SEQ ID 275 CCAGATTCATTTGGCGAGGG SEQ ID 276 AGGCTCGAAGTTCTCAACCC SEQ ID 277 CGTTAGAACTTCTACCTCTTGCC SEQ ID 278 ACCACTTCCTACACCAAATATCCC SEQ ID 279 TAATGGCAGATCCTCCACCG SEQ ID 280 ACGAATTTAAACAACAGGAGCG SEQ ID 281 CTTGTGCTTTTCAGTATACATTGGG SEQ ID 282 AATCAACTTAGCTTCCAAAGAACGC SEQ ID 283 TCAGGAAGACTAGGCATCCC SEQ ID 284 TCTTGGAGGTTATGGGTAGGG SEQ ID 285 GGGAAGCCCTTCAGAAAGAGG SEQ ID 286 CCCATTAGCGATTGCAGCTTC SEQ ID 287 CAGGACCCCATCATTCTCGC SEQ ID 288 AGCAGCTCTTACGATTGACCC SEQ ID 289 CAGGACCCCATCATTCTCGC SEQ ID 290 AGCAGCTCTTACGATTGACCC SEQ ID 291 CTCTGCTCACAAAACCAGGC SEQ ID 292 TGTATAATCTCCCTTCAGCACCC SEQ ID 293 TGAAGTGGCTCGAAGAAGCG SEQ ID 294 AGACGTTTGAATGGCTCACCC SEQ ID 295 AGATGTGTTAGAGAACAGCGC SEQ ID 296 AAGGACGATGAACAGATGCG SEQ ID 297 GGTTTTGCACCGATAAGGC SEQ ID 298 ACTAGATTCATTTGGTGGTGGC SEQ ID 299 TCACAGCCACTTTCAACAGC SEQ ID 300 GCTACAACCAGAGCGCTTTTT SEQ ID 301 TCTTCAGATTTAAGCTCATGGGGA SEQ ID 302 TGGAGGAATTTGTAAATCTTGAGACC SEQ ID 303 GTATTCTTCATGTGGCCATCCC SEQ ID 304 TGGAGAGAGGTTTTGGTTGGG SEQ ID 305 TTACTTGTGTTTGGCTGGCC SEQ ID 306 TCTGATCAGAAAATGGGAACCCG SEQ ID 307 TCGATTGGTGTTGCTAGTACGG SEQ ID 308 ACTTCCCAAAGGCTCATCCC SEQ ID 309 CGTGGAATTGAACTTCTTGGC SEQ ID 310 agCTATTTATCATCCATGCTGCC SEQ ID 311 GCTAACATGATGTTGGTCGCC SEQ ID 312 GGAATTGCTTTGGTACGAGCC SEQ ID 313 TCGATACTCGGTGATTAATccc SEQ ID 314 TTGAGCTGAGATTGAGTATAAGGC SEQ ID 315 TTCCTAACTCTTCTTCCAGTTCGC SEQ ID 316 actttttgcgAGCAAGACCG SEQ ID 317 AGCAGgacattttcaataactAATCCA SEQ ID 318 GCTCCTTACGTTCATCCAACAC SEQ ID 319 gcattgaaagaaatggaCCAAACC SEQ ID 320 TTCCAATCTAGGTCCGCAGTG SEQ ID 321 TCGTAAATGTTCTTCACCATCGAA SEQ ID 322 GAGTGGAGGTGGTTCGACTG SEQ ID 323 TCCGGTTTAAGGCGTTTCCG SEQ ID 324 TGCCAGTTCTGAATGGTACGG SEQ ID 325 GTAAGTTCCGCAATAACGTCGG SEQ ID 326 GGCGACATACGGAAATAGCC SEQ ID 327 AAGCTCAGGTGAGAACATCCC SEQ ID 328 CAGACAAGATGGGGATGGGG SEQ ID 329 CAAACCATAACCGCCATCGC SEQ ID 330 CGATGGCGTGCATTTTCCC SEQ ID 331 GATCACCAAATGCGTACAGGC SEQ ID 332 TGTTAGCGCAAGTACCTGCC SEQ ID 333 GTCTTGGGTTGATTCACTCGG SEQ ID 334 AATCCTTGAACCCAGTCGCC SEQ ID 335 GAATGCTAGACACCACAAAGGG SEQ ID 336 GTGGTTCCTTAATGCTCTCGC SEQ ID 337 AGAGACCAAACCTGCAACCC SEQ ID 338 TGGATGTCCTCTCTAGGGGG SEQ ID 339 CCAAGACAACAGAGGACCGG SEQ ID 340 TCAGAACTGTTGACATCTGGGG SEQ ID 341 TGAAATCTATTGACCCCGCCC SEQ ID 342 AATAAGGTGGTCTCCTGCCC .

[0026] The following is further described by means of specific examples.

[0027] Embodiment 1: Use standard nucleic acid to test Helicobacter pylori, Toxoplasma, Herpes virus type I, and Mycoplasma. Each sample is input with 10,000 copies. Add 4 samples to the reaction volume: name Volume μl 4* Amplification buffer 5 Primer pool mix 2 DNA 1 NF Water 12 .

[0028] Place the reaction system in a PCR instrument for amplification: .

[0029] Purification using magnetic beads: Add 25 μl of DNA purification magnetic beads for magnetic bead purification to remove small fragment dimers, and use 50 μl of water for elution.

[0030] The library was constructed using a DNA library construction kit, the kit being the Hieff NGS® UltimaPro DNA Library Prep Kit produced by Yishen Biotechnology, with the catalog number 12197.

[0031] The database construction process is: name Volume (μL) PCR amplification of DNA 50 Repair tailing buffer 6 Repair tailing enzyme 4 .

[0032] Place the reaction system in a PCR instrument for amplification: temperature time Number of cycles 30℃ 20min 1 72°C 20 min 1 4°C Hold - .

[0033] Add the connection connector: name Volume (μL) Repaired DNA 60 T4 DNA Ligation Buffer 30 DNA Adapter 5 T4 DNA Ligase 5 .

[0034] Place the reaction system in the PCR instrument and incubate: temperature time 20 °C 15 min 4 °C Hold Magnetic bead purification: Add 60ul of DNA purification magnetic beads for magnetic bead purification and use 20μL of water for elution.

[0035] PCR amplification and enrichment of the purified product: name Volume (μL) Purified DNA 20 Amplification MIX 25 Amplification primers 5 .

[0036] The amplification primers are primers with indexes, numbered 001-004. Place the reaction system in a PCR instrument and perform amplification: .

[0037] The amplified product was purified by magnetic beads, the purified product was sequenced by SE50, and the fastq file after sequencing was compared and analyzed. The results are as follows: Sample 1 Helicobacter pylori: Sample1 Detected species Offline reads 961735 Aligning reads 950421 Helicobacter pylori Hit rate 98.8% Sample 2 Toxoplasma gondii: Sample1 Detected species Offline reads 472365 Aligning reads 452749 Toxoplasma gondii Hit rate 95.8% Sample 3 Mycoplasma: Sample1 Detected species Offline reads 873214 Aligning reads 856487 Mycoplasma Hit rate 98.1% Sample 4 Feline Herpesvirus Type 1: Sample1 Detected species Offline reads 781574 Aligning reads 778943 Feline herpesvirus type I Hit rate 99.6% This example shows that in the process of single-species detection, different targets can specifically amplify the target region, and the target rate is greater than 95%. Specific embodiment 2: Using standard nucleic acid, 10,000 copies of Helicobacter pylori, 1,000 copies of Mycoplasma, 100 copies of Toxoplasma, and 10 copies of Feline Herpesvirus Type I were mixed and added to the reaction system: name Volume μl 4* Amplification buffer 5 Primer pool mix 2 DNA 4 NF Water 9 .

[0039] Make a tube of NC in parallel as a control: name Volume μl 4* Amplification buffer 5 Primer pool mix 2 NC DNA 1 NF Water 13 .

[0040] Place the reaction system in a PCR instrument for amplification: .

[0041] Purification using magnetic beads: Add 25 μl of DNA purification magnetic beads for magnetic bead purification to remove small fragment dimers, and use 50 μl of water for elution.

[0042] The library was constructed using a DNA library construction kit, the kit being the Hieff NGS® UltimaPro DNA Library Prep Kit produced by Yishen Biotechnology, with the catalog number 12197.

[0043] The database construction process is: name Volume (μL) PCR amplification of DNA 50 Repair tailing buffer 6 Repair tailing enzyme 4 .

[0044] Place the reaction system in a PCR instrument for amplification: temperature time Number of cycles 30℃ 20min 1 72°C 20 min 1 4°C Hold - .

[0045] Add the connection connector: name Volume (μL) Repaired DNA 60 T4 DNA Ligation Buffer 30 DNA Adapter 5 T4 DNA Ligase 5 .

[0046] Place the reaction system in the PCR instrument and incubate: temperature time 20 °C 15 min 4 °C Hold Magnetic bead purification: Add 60ul of DNA purification magnetic beads for magnetic bead purification and use 20μL of water for elution.

[0047] PCR amplification and enrichment of the purified product: name Volume (μL) Purified DNA 20 Amplification MIX 25 Amplification primers 5 The amplification primers are primers with indexes, numbered 005 and 006.

[0048] Place the reaction system in a PCR instrument for amplification: .

[0049] The amplified product was purified by magnetic beads, the purified product was sequenced by SE50, and the fastq file after sequencing was compared and analyzed. The results are as follows: Checkout sample1 sample2 Helicobacter pylori 578902 23 Mycoplasma 48734 5 Toxoplasma gondii 3578 0 Feline herpesvirus type I 747 0 NC 24 278134 .

[0050] The positive standard in the judgment sample showed strong linearity from 10,000 copies to 10 copies, which was basically consistent with the spike-in ratio. At the same time, due to experimental and sequencing reasons, the same batch of controls had a small amount of contamination. The same batch of controls were also detected in the positive samples.

Claims

1. A single-tube multiplex PCR pet pathogen detection kit, comprising an amplification buffer, an end repair buffer, a connection adapter, a library amplification primer and an amplification enzyme, characterized in that: Specific regions of 34 species were screened, and a set of internal reference genes was prepared. Based on this, upstream and downstream primers were designed, including primer sets 1~342, which can simultaneously detect Lyme disease, Helicobacter pylori, Bordetella, Chlamydia, Ehrlichia, Anaplasma, Mycoplasma, Giardia, Toxoplasma, Heartworm, Feline Herpesvirus Type I, Canine Adenovirus Type I, Canine Adenovirus Type II, Feline Parvovirus, Canine Parvovirus, Canine A Coronavirus Type I, Rabies Virus, Feline Immunodeficiency Virus, Feline Leukemia Virus, Feline Calicivirus, Feline A Coronavirus Type I, Haemophilus Mycoplasma, Canine Babesia, Bartonella henselae, Rickettsia, Bartonella clarridgeae, Rickettsia, Leptospira, Feline Cryptosporidium, Eperythrozoon, Canine Parainfluenza Virus, Canine Cryptosporidium, Tritrichomonas fetus, and Canine Distemper Virus.

2. A single-tube multiplex PCR pet pathogen detection kit according to claim 1, characterized in that: Primers SEQ ID 1 to 10 are Lyme disease-specific primers; Primers SEQ ID 11 to 20 are Helicobacter pylori primers; Primers SEQ ID 21 to 30 are Bordetella; Primers SEQ ID 31 to 40 are Chlamydia; Primers SEQ ID 41 to 50 are for Ehrlichia; Primers SEQ ID 51 to 60 are for Anaplasma; Primers SEQ ID 61 to 70 are Mycoplasma; Primers SEQ ID 71-80 are Giardia lamblia; Primers SEQ ID 81 to 90 are Toxoplasma gondii; Primers SEQ ID 91 to 100 are Heartworm; Primers SEQ ID 101-110 are feline herpesvirus type I; Primers SEQ ID 111-110 are canine adenovirus type I; Primers SEQID 121 to 130 are canine adenovirus type II; Primers SEQ ID 131-140 are feline parvovirus; Primers SEQ ID 141-150 are canine parvovirus; Primers SEQ ID 151-158 are canine alpha coronavirus type I; Primers SEQ ID 159-168 are rabies virus; Primers SEQ ID 169-178 are feline immunodeficiency virus; Primers SEQ ID 179-188 are feline leukemia virus; Primers SEQ ID 189-196 are feline calicivirus; Primers SEQ ID 197-206 are feline alpha coronavirus type I; Primers SEQ ID 207-216 are Haemophilus influenzae; Primers SEQ ID 217 to 226 are for Babesia canis; Primers SEQ ID 227 to 236 are for Bartonella henselae; Primers SEQ ID 237-246 are for Rickettsia; Primers SEQ ID 247-256 are for Bartonella clarridgeae; Primers SEQ ID 257-266 are for Rickettsia; Primers SEQ ID 267-276 are for Leptospira; Primers SEQ ID 277-286 are for Cryptosporidium felis; Primers SEQ ID 287-296 are Eperythrozoon; Primers SEQ ID 297-301 are Canine Parainfluenza Virus; Primers SEQ ID 303-311 are Cryptosporidium canis; Primers SEQ ID 313-322 are Tritrichomonas foetus; Primers SEQ ID 323-332 are canine distemper virus; primers SEQ ID 333-342 are internal reference genes.

3. A single-tube multiplex PCR pet pathogen detection kit according to claim 1, characterized in that: Amplification buffer and primer pool were mixed, nucleic acid was added, and amplification was performed. After the amplification product was purified, the library construction kit was used for end repair, adapter ligation, and library enrichment amplification.

4. According to claim 3, the final concentration of the multiple amplification buffer system is 10~30 mM Tris-HCl, 60~100 mM KCl, 10~30 mM (NH4)2SO4, 0.5~2.0 mM MgCl2, 0.1~0.2 mM dNTPs, 5~10% glycerol, 0.05~0.1% Tween® 205~10% polyethylene glycol (PEG), 1~5% dimethyl sulfoxide (DMSO), 0.2~2U Taq DNA Polymerase, pH 8.5~9.0.