CN114058736A - Pig pathogenic bacteria detection method - Google Patents

Pig pathogenic bacteria detection method Download PDF

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CN114058736A
CN114058736A CN202110867974.XA CN202110867974A CN114058736A CN 114058736 A CN114058736 A CN 114058736A CN 202110867974 A CN202110867974 A CN 202110867974A CN 114058736 A CN114058736 A CN 114058736A
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primer
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郭村勇
陈灿坚
谢旺儒
陈台安
李柏宽
谢凯如
江曜安
萧志奇
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Advanced Application Technology Co ltd
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Abstract

The invention relates to a method for detecting pig pathogenic bacteria. In addition, the invention also relates to an oligonucleotide pair for detecting the pig pathogenic bacteria.

Description

Pig pathogenic bacteria detection method
Technical Field
The present invention relates to a method for detecting porcine pathogenic bacteria, and more particularly to a method for detecting porcine pathogenic bacteria using oligonucleotides.
Background
Pigs are one of the most important economic animals worldwide. According to the statistics of the United states department of agriculture, the total pork yield of the major pig-raising countries in the world in 2018 reaches 113,081 kilotons. The economic value of pork is over billions of dollars. However, intensive farming coupled with poor management may result in pigs that are only susceptible to disease and the epidemic spreads rapidly. Taking the outbreak of African swine fever in 2018 as an example, the death rate of sick pigs is almost 100%, and huge economic losses are caused. It follows that intensive monitoring of pigs in the farm is an indispensable part of management. The invention provides a rapid and convenient pig pathogenic bacteria detection method for industrial utilization.
Disclosure of Invention
In one aspect, the present invention relates to a swine pathogen detection method, comprising providing a sample that may contain one or more nucleotide sequences of a swine pathogen; providing an oligonucleotide primer pair defining the 5' ends of two complementary strands of a double stranded target sequence on one or more nucleotide sequences of the pathogen, the oligonucleotide primer pair comprising a first primer and a second primer; providing a polymerase; mixing the sample, the pair of oligonucleotide primers, the polymerase, deoxyadenosine triphosphates (dATPs), deoxycytidine triphosphates (dCTPs), deoxyguanosine triphosphates (dGTPs), and deoxythymidine triphosphates (dTTPs) in a container to form a Polymerase Chain Reaction (PCR) mixture; subjecting the PCR mixture to a thermal convection polymerase chain reaction (cPCR) by heating the bottom of the vessel at a fixed temperature to form a PCR product; and detecting the PCR product to identify the double-stranded target sequence.
In certain embodiments, the pathogen is Classical Swine Fever Virus (CSFV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO. 1 and 7, SEQ ID NO. 2 and 8, SEQ ID NO. 5 and 8, SEQ ID NO. 3 and 9, SEQ ID NO. 4 and 10, SEQ ID NO. 4 and 11, SEQ ID NO. 6 and 10, and SEQ ID NO. 6 and 11.
In certain embodiments, the pathogenic bacterium is Foot and Mouth Disease Virus (FMDV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 16 and 22 SEQ ID NO, 17 and 23 SEQ ID NO, 20 and 23 SEQ ID NO, 18 and 24 SEQ ID NO, 20 and 24 SEQ ID NO, 19 and 25 SEQ ID NO, 19 and 26 SEQ ID NO, 21 and 25 SEQ ID NO, and 21 and 26 SEQ ID NO.
In certain embodiments, the pathogenic bacterium is a Japanese Encephalitis Virus (JEV), and the sequence combination of the first primer and the second primer is selected from the following combinations: 31 and 35 of SEQ ID NO, 31 and 36 of SEQ ID NO, 32 and 35 of SEQ ID NO, 33 and 36 of SEQ ID NO, 34 and 35 of SEQ ID NO, and 34 and 36 of SEQ ID NO.
In certain embodiments, the pathogenic bacteria is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO. 40 and SEQ ID NO. 44, SEQ ID NO. 43 and SEQ ID NO. 44, SEQ ID NO. 40 and SEQ ID NO. 45, SEQ ID NO. 41 and SEQ ID NO. 47, and SEQ ID NO. 42 and SEQ ID NO. 46.
In certain embodiments, the pathogenic bacterium is Swine influenza a virus (SIV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 51 and SEQ ID NO 56, SEQ ID NO 54 and SEQ ID NO 56, SEQ ID NO 51 and SEQ ID NO 57, SEQ ID NO 52 and SEQ ID NO 56, SEQ ID NO 55 and SEQ ID NO 56, and SEQ ID NO 53 and SEQ ID NO 56.
In certain embodiments, the pathogenic bacterium is Porcine Epidemic Diarrheal Virus (PEDV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 61 and 67, SEQ ID NO 62 and 68, SEQ ID NO 65 and 68, SEQ ID NO 63 and 69, SEQ ID NO 63 and 71, SEQ ID NO 64 and 70, SEQ ID NO 66 and 70, and SEQ ID NO 66 and 72.
In certain embodiments, the pathogen is Porcine circovirus type 2 (PCV2) and the sequence combination of the first primer and the second primer is selected from the group consisting of: 77 and 82 SEQ ID NO, 78 and 83 SEQ ID NO, 79 and 84 SEQ ID NO, 80 and 85 SEQ ID NO, and 81 and 84 SEQ ID NO.
In certain embodiments, the pathogenic bacterium is Pseudorabies virus (PRV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO. 89 and 96, SEQ ID NO. 90 and 97, SEQ ID NO. 91 and 98, SEQ ID NO. 92 and 99, SEQ ID NO. 93 and 100, SEQ ID NO. 94 and 101, and SEQ ID NO. 95 and 102.
In certain embodiments, the pathogenic bacterium is porcine Seneca Valley Virus A (SVVA), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 107 and 112 of SEQ ID NO, 107 and 116 of SEQ ID NO, 111 and 112 of SEQ ID NO, 111 and 116 of SEQ ID NO, 108 and 113 of SEQ ID NO, 109 and 114 of SEQ ID NO, and 110 and 115 of SEQ ID NO.
In certain embodiments, the pathogen is Mycoplasma hyopneumoniae (Mhp), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 121 and 126, SEQ ID NO 121 and 130, SEQ ID NO 125 and 126, SEQ ID NO 125 and 130, SEQ ID NO 122 and 127, SEQ ID NO 123 and 128, and SEQ ID NO 124 and 129.
In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe comprising a sequence complementary to a segment of the double-stranded target sequence, a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe, the fluorescence-inhibiting molecule substantially inhibiting the fluorescent molecule when the oligonucleotide probe is not hybridized to the segment of the double-stranded target sequence and the fluorescent molecule is not substantially inhibited when the oligonucleotide probe is hybridized to the segment of the double-stranded target sequence.
In certain embodiments, the pathogen is Classical Swine Fever Virus (CSFV), and the oligonucleotide primer pair comprises a first primer and a second primer, the first primer having a sequence as set forth in SEQ ID NO. 1 and the second primer having a sequence as set forth in SEQ ID NO. 7. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is a 13 to 30 base pair oligonucleotide between the 147 th to 190 th nucleotides of the complete genomic sequence (GenBank access No. af531433) of the pathogenic bacterium Classical Swine Fever Virus (CSFV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 12.
In certain embodiments, the pathogen is Classical Swine Fever Virus (CSFV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 2 and 8, and 5 and 8. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between nucleotides 6482 to 6515 of the complete genomic sequence (GenBank access No. af531433) of the pathogenic bacterium Classical Swine Fever Virus (CSFV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 13.
In certain embodiments, the pathogen is Classical Swine Fever Virus (CSFV), and the sequence of the first primer is set forth in SEQ ID NO. 3 and the sequence of the second primer is set forth in SEQ ID NO. 9. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is a 13 to 30 base pair oligonucleotide between nucleotides 175 to 219 of the complete genomic sequence (GenBank access No. af531433) of the pathogenic bacterium Classical Swine Fever Virus (CSFV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO. 14.
In certain embodiments, the pathogen is Classical Swine Fever Virus (CSFV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO. 4 and 10, SEQ ID NO. 4 and 11, SEQ ID NO. 6 and 10, and SEQ ID NO. 6 and 11. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is a 13 to 30 base pair oligonucleotide between the 139 th to 170 th nucleotides of the complete genomic sequence (GenBank access No. af531433) of the pathogenic bacterium Classical Swine Fever Virus (CSFV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO. 15.
In certain embodiments, the pathogenic bacterium is Foot and Mouth Disease Virus (FMDV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 16 and 22, 17 and 23, 20 and 23, 18 and 24, and 20 and 24. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between nucleotides 7907 to 7949 of the complete genomic sequence (GenBank access No. mf461724.1) of the pathogenic Foot and Mouth Disease Virus (FMDV). In certain preferred embodiments, the sequence of the oligonucleotide probe is selected from any one of SEQ ID NOs 27, 28 and 29.
In certain embodiments, the pathogenic bacterium is foot-and-mouth disease virus (FMDV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 19 and 25 SEQ ID NO, 19 and 26 SEQ ID NO, 21 and 25 SEQ ID NO, and 21 and 26 SEQ ID NO. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between the 8003 to 8031 nucleotides of the complete genomic sequence (GenBank access No. mf461724.1) of the pathogenic Foot and Mouth Disease Virus (FMDV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 30.
In certain embodiments, the pathogenic bacterium is a Japanese Encephalitis Virus (JEV), and the sequence combination of the first primer and the second primer is selected from the following combinations: 31 and 35 of SEQ ID NO, 31 and 36 of SEQ ID NO, 32 and 35 of SEQ ID NO, 33 and 36 of SEQ ID NO, 34 and 35 of SEQ ID NO, and 34 and 36 of SEQ ID NO. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is a 13 to 25 base pair oligonucleotide between the 3599 to 3626 nucleotides of the complete genomic sequence (GenBank access No. ab051292) of the pathogenic JEV. In certain preferred embodiments, the sequence of the oligonucleotide probe is selected from any one of SEQ ID NOs 37, 38 and 39.
In certain embodiments, the pathogenic bacteria is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 40 and 44, and 43 and 44. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between 14532 th to 14554 th nucleotides of the complete genomic sequence (GenBank access No. ef536003) of the pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO 48.
In certain embodiments, the pathogenic bacterium is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 40 and 45 of SEQ ID NO, 41 and 45 of SEQ ID NO, and 41 and 47 of SEQ ID NO. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between 14553 to 14584 nucleotides of the complete genomic sequence (GenBank access No. ef536003) of the pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO. 49.
In certain embodiments, the pathogen is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and the first primer has the sequence shown in SEQ ID NO. 42 and the second primer has the sequence shown in SEQ ID NO. 46. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between 14501 to 14537 nucleotides of the complete genomic sequence (GenBank access No. ef536003) of the pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO 50.
In certain embodiments, the pathogenic bacterium is Swine influenza a virus (SIV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 51 and 56, SEQ ID NO 54 and 56, and SEQ ID NO 51 and 57. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between nucleotides 205 to 237 of the Matrix Protein 1(Matrix Protein1) sequence (GenBank access No. mh234432) of the pathogenic bacterium swine influenza a virus (SIV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO: 58.
In certain embodiments, the pathogenic bacterium is swine influenza a virus (SIV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: 52 and 56, and 55 and 56. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between the 200 th to 239 th nucleotides of the Matrix Protein 1(Matrix Protein1) sequence (GenBank access No. mh234432) of the pathogenic bacterium swine influenza a virus (SIV). In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO 59.
In certain embodiments, the pathogen is swine influenza A virus (SIV) and the sequence of the first primer is set forth in SEQ ID NO:53 and the sequence of the second primer is set forth in SEQ ID NO: 56. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between the 169 th to 239 rd nucleotides of the Matrix Protein 1(Matrix Protein1) sequence (GenBank access No. mh234432) of the pathogenic bacterium swine influenza a virus (SIV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 60.
In certain embodiments, the pathogen is swine influenza a virus (SIV), and the sample source is not limited to swine, but may also be derived from a canine.
In certain embodiments, the pathogen is Porcine Epidemic Diarrheal Virus (PEDV), and the sequence of the first primer is set forth in SEQ ID NO:61 and the sequence of the second primer is set forth in SEQ ID NO: 67. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is a 13 to 30 base pair oligonucleotide between 156 th to 212 th nucleotides of the complete genomic sequence (GenBank access No. af353511) of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO. 73.
In certain embodiments, the pathogenic bacterium is Porcine Epidemic Diarrhea Virus (PEDV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 62 and 68, and SEQ ID NO 65 and 68. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between the 27189 th to 27218 th nucleotides of the complete genomic sequence (GenBank access No. af353511) of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 74.
In certain embodiments, the pathogenic bacterium is Porcine Epidemic Diarrhea Virus (PEDV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 63 and 69, and SEQ ID NO 63 and 71. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between the 26588 th to 26625 th nucleotides of the complete genomic sequence (GenBank access No. af353511) of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV). In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO 75.
In certain embodiments, the pathogenic bacterium is Porcine Epidemic Diarrhea Virus (PEDV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 64 and 70, SEQ ID NO 66 and 70, and SEQ ID NO 66 and 72. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is a 13 to 30 base pair oligonucleotide between the 22431 st to the 22454 th nucleotides of the complete genomic sequence of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV) (GenBank access No. af353511). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO. 76.
In certain embodiments, the pathogen is Porcine circovirus type 2 (PCV2) and the sequence combination of the first primer and the second primer is selected from the group consisting of: 77 and 82, 78 and 82, and 78 and 83. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between the 66 th to 94 th nucleotides of the complete genomic sequence (GenBank access No. km487709) of the pathogenic porcine circovirus type 2 (PCV 2). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 86.
In certain embodiments, the pathogen is porcine circovirus type 2 (PCV2) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 79 and 84, and SEQ ID NO 81 and 84. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is a 13 to 30 base pair oligonucleotide between the 523 to 584 nucleotides of the complete genomic sequence (GenBank access No. km487709) of the pathogenic porcine circovirus type 2 (PCV 2). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 87.
In certain embodiments, the pathogen is porcine circovirus type 2 (PCV2), and the sequence of the first primer is set forth in SEQ ID NO:80 and the sequence of the second primer is set forth in SEQ ID NO: 85. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between the 487 to 546 nucleotides of the complete genomic sequence (GenBank access No. km487709) of the pathogenic porcine circovirus type 2 (PCV 2). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO: 88.
In certain embodiments, the pathogen is Pseudorabies virus (PRV), and the sequence of the first primer is set forth in SEQ ID NO. 89 and the sequence of the second primer is set forth in SEQ ID NO. 96. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 25 base pairs between 17998 to 18024 nucleotides of the complete genomic sequence (GenBank access No. jf797217.1) of the pathogenic pseudorabies virus (PRV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO. 103.
In certain embodiments, the pathogenic bacterium is pseudorabies virus (PRV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 90 and 97, and SEQ ID NO 91 and 98. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between nucleotides 18244 to 18298 of the complete genomic sequence of the pathogenic pseudorabies virus (PRV) (GenBank access No. jf797217.1). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 104.
In certain embodiments, the pathogenic bacterium is pseudorabies virus (PRV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 92 and 99, and SEQ ID NO 93 and 100. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 25 base pairs between nucleotides 16204 to 16225 of the complete genomic sequence (GenBank access No. jf797217.1) of the pathogenic pseudorabies virus (PRV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 105.
In certain embodiments, the pathogenic bacterium is pseudorabies virus (PRV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: 94 and 101, and 95 and 102. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between 18000 to 18030 nucleotides of the complete genomic sequence (GenBank access No. jf797217.1) of the pathogenic pseudorabies virus (PRV). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 106.
In certain embodiments, the pathogenic bacterium is porcine Seneca Valley Virus A (SVVA) and the sequence combination of the first primer and the second primer is selected from the group consisting of: 107 and 112 of SEQ ID NO, 107 and 116 of SEQ ID NO, 111 and 112 of SEQ ID NO, and 111 and 116 of SEQ ID NO. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 25 base pairs between the 7054 th to 7079 th nucleotides of the complete genomic sequence (GenBank access No. dq641257.1) of the pathogenic porcine Sendai virus (SVVA). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 117.
In certain embodiments, the pathogen is porcine Sambucus Valley Virus (SVVA) and the sequence of the first primer is set forth in SEQ ID NO:108 and the sequence of the second primer is set forth in SEQ ID NO: 113. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 17 base pairs between 6933 to 6949 nucleotides of the complete genomic sequence (GenBank accession No. dq641257.1) of the pathogenic porcine Sendai Valley Virus (SVVA). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 118.
In certain embodiments, the pathogen is porcine Sambucus Valley Virus (SVVA) and the first primer has the sequence shown in SEQ ID NO. 109 and the second primer has the sequence shown in SEQ ID NO. 114. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 21 base pairs between the 6953 rd to 6973 rd nucleotides of the complete genomic sequence (GenBank access No. dq641257.1) of the pathogenic porcine Sendai virus (SVVA). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 119.
In certain embodiments, the pathogen is porcine Sambucus Valley Virus (SVVA) and the first primer has the sequence shown in SEQ ID NO. 110 and the second primer has the sequence shown in SEQ ID NO. 115. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 21 base pairs between 7154 to 7174 nucleotides of the complete genomic sequence (GenBank access No. dq641257.1) of the pathogenic porcine s-agallovirus (SVVA). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 120.
In certain embodiments, the pathogen is Mycoplasma hyopneumoniae (Mhp), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 121 and 126, 121 and 130, 125 and 126, and 125 and 130. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between the 215406 th to 215441 th nucleotides of the complete genomic sequence (GenBank access No. ae017243.1) of the pathogenic mycoplasma hyopneumoniae (Mhp). In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO 131.
In certain embodiments, the pathogen is Mycoplasma hyopneumoniae (Mhp), and the sequence of the first primer is set forth in SEQ ID NO:122 and the sequence of the second primer is set forth in SEQ ID NO: 127. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between the 215742 th to 215772 th nucleotides of the complete genomic sequence (GenBank access No. ae017243.1) of the pathogenic mycoplasma hyopneumoniae (Mhp). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO: 132.
In certain embodiments, the pathogen is Mycoplasma hyopneumoniae (Mhp) and the sequence of the first primer is set forth in SEQ ID NO 123 and the sequence of the second primer is set forth in SEQ ID NO 128. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 25 base pairs between the 215750 th to 215774 th nucleotides of the complete genomic sequence (GenBank access No. ae017243.1) of the pathogenic mycoplasma hyopneumoniae (Mhp). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 133.
In certain embodiments, the pathogen is Mycoplasma hyopneumoniae (Mhp) and the sequence of the first primer is set forth in SEQ ID NO:124 and the sequence of the second primer is set forth in SEQ ID NO: 129. In certain embodiments, the Polymerase Chain Reaction (PCR) mixture further comprises an oligonucleotide probe that is an oligonucleotide of 13 to 30 base pairs between the 215750 th to 215780 th nucleotides of the complete genomic sequence (GenBank access No. ae017243.1) of the pathogenic mycoplasma hyopneumoniae (Mhp). In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 134.
In one aspect, the invention relates to an oligonucleotide pair for detecting porcine pathogens, comprising a first primer and a second primer.
In certain embodiments, the pathogen is Classical Swine Fever Virus (CSFV), and the sequence of the first primer is set forth in SEQ ID NO. 1 and the sequence of the second primer is set forth in SEQ ID NO. 2. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between nucleotides 147 to 190 of the complete genomic sequence (GenBank access No. af531433) of the pathogenic bacterium Classical Swine Fever Virus (CSFV), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 12.
In certain embodiments, the pathogen is Classical Swine Fever Virus (CSFV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: 2 and 8, and 5 and 8. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between nucleotides 6482 to 6515 of the complete genomic sequence of pathogenic Classical Swine Fever Virus (CSFV) (GenBank access No. af531433), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 13.
In certain embodiments, the pathogen is Classical Swine Fever Virus (CSFV), and the sequence of the first primer is set forth in SEQ ID NO. 3 and the sequence of the second primer is set forth in SEQ ID NO. 9. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between nucleotides 175 to 219 of the complete genomic sequence (GenBank access No. af531433) of the pathogenic bacterium Classical Swine Fever Virus (CSFV), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO. 14.
In certain embodiments, the pathogen is Classical Swine Fever Virus (CSFV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO. 4 and 10, SEQ ID NO. 4 and 11, SEQ ID NO. 6 and 10, and SEQ ID NO. 6 and 11. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between nucleotides 139 to 170 of the complete genomic sequence (GenBank access No. af531433) of the pathogenic bacterium Classical Swine Fever Virus (CSFV), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO. 15.
In certain embodiments, the pathogenic bacterium is foot-and-mouth disease virus (FMDV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 16 and 22, 17 and 23, 20 and 23, 18 and 24, and 20 and 24. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between nucleotides 7907 to 7949 of the complete genomic sequence (GenBank access No. mf461724.1) of the pathogenic Foot and Mouth Disease Virus (FMDV), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the sequence of the oligonucleotide probe is selected from any one of SEQ ID NOs 27, 28 and 29.
In certain embodiments, the pathogenic bacterium is foot-and-mouth disease virus (FMDV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 19 and 25 SEQ ID NO, 19 and 26 SEQ ID NO, 21 and 25 SEQ ID NO, and 21 and 26 SEQ ID NO. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between the 8003 to the 8031 nucleotides of the complete genomic sequence (GenBank access No. mf461724.1) of the pathogenic bacteria Foot and Mouth Disease Virus (FMDV), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 30.
In certain embodiments, the pathogenic bacterium is Japanese Encephalitis Virus (JEV), and the combination of sequences of the first and second primers is selected from the following combinations: 31 and 35 of SEQ ID NO, 31 and 36 of SEQ ID NO, 32 and 35 of SEQ ID NO, 33 and 36 of SEQ ID NO, 34 and 35 of SEQ ID NO, and 34 and 36 of SEQ ID NO. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 25 base pairs between the 3599 to 3626 nucleotides of the complete genomic sequence (GenBank access No. ab051292) of the pathogenic JEV, a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the sequence of the oligonucleotide probe is selected from any one of SEQ ID NOs 37, 38 and 39.
In certain embodiments, the pathogenic bacterium is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 40 and 44, and 43 and 44. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe that is a 13 to 30 base pair oligonucleotide between the 14532 th to 14554 th nucleotides of the complete genomic sequence (GenBank access No. ef536003) of the pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO 48.
In certain embodiments, the pathogenic bacterium is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: 40 and 45 of SEQ ID NO, 41 and 45 of SEQ ID NO, and 41 and 47 of SEQ ID NO. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe that is a 13 to 30 base pair oligonucleotide between nucleotides 14553 to 14584 of the complete genomic sequence (GenBank access No. ef536003) of the pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO. 49.
In certain embodiments, the pathogen is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the first primer has the sequence shown in SEQ ID NO:42, and the second primer has the sequence shown in SEQ ID NO: 46. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe that is a 13 to 30 base pair oligonucleotide between nucleotides 14501 to 14537 of the complete genomic sequence (GenBank access No. ef536003) of the pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO 50.
In certain embodiments, the pathogenic bacterium is swine influenza a virus (SIV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 51 and 56, SEQ ID NO 54 and 56, and SEQ ID NO 51 and 57. In certain embodiments, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between nucleotides 205 to 237 of the Matrix Protein 1(Matrix Protein1) sequence (GenBank access No. mh234432) of the pathogenic bacterium swine influenza a virus (SIV), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO: 58.
In certain embodiments, the pathogenic bacterium is swine influenza a virus (SIV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: 52 and 56, and 55 and 56. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between the 200 th to the 239 th nucleotides of the Matrix Protein 1(Matrix Protein1) sequence (GenBank access No. mh234432) of the pathogenic bacterium swine influenza a virus (SIV), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO 59.
In certain embodiments, the pathogen is swine influenza A virus (SIV) and the sequence of the first primer is set forth in SEQ ID NO:53 and the sequence of the second primer is set forth in SEQ ID NO: 56. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between nucleotides 169 to 239 of the Matrix Protein 1(Matrix Protein1) sequence (GenBank access No. mh234432) of the pathogenic bacterium swine influenza a virus (SIV), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 60.
In certain embodiments, the pathogen is Porcine Epidemic Diarrhea Virus (PEDV) and the sequence of the first primer is set forth in SEQ ID NO:61 and the sequence of the second primer is set forth in SEQ ID NO: 67. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between 156 th to 212 th nucleotides of the complete genomic sequence (GenBank access No. af353511) of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO. 73.
In certain embodiments, the pathogenic bacterium is Porcine Epidemic Diarrhea Virus (PEDV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 62 and 68, and SEQ ID NO 65 and 68. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between the 27189 th to 27218 th nucleotides of the complete genomic sequence (GenBank access No. af353511) of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 74.
In certain embodiments, the pathogenic bacterium is Porcine Epidemic Diarrhea Virus (PEDV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 63 and 69, and SEQ ID NO 63 and 71. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between the 26588 th to 26625 th nucleotides of the complete genomic sequence (GenBank access No. af353511) of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO 75.
In certain embodiments, the pathogenic bacterium is Porcine Epidemic Diarrhea Virus (PEDV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 64 and 70, SEQ ID NO 66 and 70, and SEQ ID NO 66 and 72. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between 22431 and 22454 nucleotides of the complete genomic sequence of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV) (GenBank access No. af353511), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO. 76.
In certain embodiments, the pathogen is porcine circovirus type 2 (PCV2) and the sequence combination of the first primer and the second primer is selected from the group consisting of: 77 and 82, 78 and 82, and 78 and 83. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between the 66 th to 94 th nucleotides of the complete genomic sequence (GenBank access No. km487709) of the pathogenic porcine circovirus type 2 (PCV2), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 86.
In certain embodiments, the pathogen is porcine circovirus type 2 (PCV2) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 79 and 84, and SEQ ID NO 81 and 84. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between the 523 to 584 nucleotides of the complete genomic sequence (GenBank access No. km487709) of the pathogenic porcine circovirus type 2 (PCV2), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 87.
In certain embodiments, the pathogen is porcine circovirus type 2 (PCV2), and the sequence of the first primer is set forth in SEQ ID NO:80 and the sequence of the second primer is set forth in SEQ ID NO: 85. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between the 487 to 546 nucleotides of the complete genomic sequence (GenBank access No. km487709) of the pathogenic porcine circovirus type 2 (PCV2), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO: 88.
In certain embodiments, the pathogen is pseudorabies virus (PRV) and the sequence of the first primer is set forth in SEQ ID NO. 89 and the sequence of the second primer is set forth in SEQ ID NO. 96. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 25 base pairs between 17998 to 18024 nucleotides of the complete genomic sequence (GenBank access No. jf797217.1) of the pathogenic pseudorabies virus (PRV), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO. 103.
In certain embodiments, the pathogenic bacterium is pseudorabies virus (PRV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 90 and 97, and SEQ ID NO 91 and 98. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between 18244 and 18298 nucleotides of the complete genomic sequence of the pathogenic pseudorabies virus (PRV) (GenBank access No. jf797217.1), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 104.
In certain embodiments, the pathogenic bacterium is pseudorabies virus (PRV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO 92 and 99, and SEQ ID NO 93 and 100. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 25 base pairs between nucleotides 16204 to 16225 of the complete genomic sequence of the pathogenic pseudorabies virus (PRV) (GenBank access No. jf797217.1), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 105.
In certain embodiments, the pathogenic bacterium is pseudorabies virus (PRV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: 94 and 101, and 95 and 102. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 25 base pairs between nucleotides 18000 to 18030 of the complete genomic sequence (GenBank access No. jf797217.1) of the pathogenic pseudorabies virus (PRV), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 106.
In certain embodiments, the pathogenic bacterium is porcine Sendai Valley Virus (SVVA) and the sequence combination of the first primer and the second primer is selected from the group consisting of: 107 and 112 of SEQ ID NO, 107 and 116 of SEQ ID NO, 111 and 112 of SEQ ID NO, and 111 and 116 of SEQ ID NO. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 25 base pairs between the 7054 th to 7079 th nucleotides of the complete genomic sequence (GenBank access No. dq641257.1) of the pathogenic porcine s-agallovirus (SVVA), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 117.
In certain embodiments, the pathogen is porcine Sambucus Valley Virus (SVVA) and the sequence of the first primer is set forth in SEQ ID NO:108 and the sequence of the second primer is set forth in SEQ ID NO: 113. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 17 base pairs between 6933 and 6949 nucleotides of the complete genomic sequence (GenBank accession No. dq641257.1) of the pathogenic porcine s-agallovirus (SVVA), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 118.
In certain embodiments, the pathogen is porcine Sambucus Valley Virus (SVVA) and the first primer has the sequence shown in SEQ ID NO. 109 and the second primer has the sequence shown in SEQ ID NO. 114. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 21 base pairs between the 6953 rd to 6973 rd nucleotides of the complete genomic sequence (GenBank access No. dq641257.1) of the pathogenic porcine s-agallovirus (SVVA), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 119.
In certain embodiments, the pathogen is porcine Sambucus Valley Virus (SVVA) and the first primer has the sequence shown in SEQ ID NO. 110 and the second primer has the sequence shown in SEQ ID NO. 115. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 21 base pairs between 7154 and 7174 nucleotides of the complete genomic sequence (GenBank access No. dq641257.1) of the pathogenic porcine s-agallovirus (SVVA), a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 120.
In certain embodiments, the pathogen is mycoplasma hyopneumoniae (Mhp) and the sequence combination of the first primer and the second primer is selected from the group consisting of: 121 and 126, 121 and 130, 125 and 126, and 125 and 130. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between the 215406 th to 215441 th nucleotides of the complete genomic sequence (GenBank access No. ae017243.1) of the pathogenic mycoplasma hyopneumoniae (Mhp), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has a sequence as set forth in SEQ ID NO 131.
In certain embodiments, the pathogen is Mycoplasma hyopneumoniae (Mhp), and the sequence of the first primer is set forth in SEQ ID NO:122 and the sequence of the second primer is set forth in SEQ ID NO: 127. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between the 215742 th to 215772 th nucleotides of the complete genomic sequence (GenBank access No. ae017243.1) of the pathogenic mycoplasma hyopneumoniae (Mhp), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO: 132.
In certain embodiments, the pathogen is Mycoplasma hyopneumoniae (Mhp), and the sequence of the first primer is set forth in SEQ ID NO:123 and the sequence of the second primer is set forth in SEQ ID NO: 128. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 25 base pairs between the 215750 th to 215774 th nucleotides of the complete genomic sequence (GenBank access No. ae017243.1) of the pathogenic mycoplasma hyopneumoniae (Mhp), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 133.
In certain embodiments, the pathogen is Mycoplasma hyopneumoniae (Mhp), and the sequence of the first primer is set forth in SEQ ID NO:124 and the sequence of the second primer is set forth in SEQ ID NO: 129. In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe comprising an oligonucleotide of 13 to 30 base pairs between the 215750 th to 215780 th nucleotides of the complete genomic sequence (GenBank access No. ae017243.1) of the pathogenic mycoplasma hyopneumoniae (Mhp), a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second location on the oligonucleotide probe. In certain preferred embodiments, the oligonucleotide probe has the sequence shown in SEQ ID NO 134.
Drawings
FIG. 1 shows the results of real-time PCR (real-time PCR) using primers CSFV-F1 and CSFV-R1 and probe CSFV-P1 for Classical Swine Fever Virus (CSFV).
FIG. 2 shows the results of real-time PCR (real-time PCR) detection of foot-and-mouth disease virus (FMDV) using primer FMDV-F1 and FMDV-R1 and probe FMDV-P1.
FIG. 3 shows the results of real-time PCR (real-time PCR) for detecting Japanese Encephalitis Virus (JEV) using primers JEV-F4 and JEV-R1 and probe JEV-P1.
FIG. 4 shows the results of real-time PCR (real-time PCR) detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using primers PRRSV-F1 and PRRSV-R2 and probe PRRSV-P2.
FIG. 5 shows the results of real-time PCR (real-time PCR) for detecting swine influenza A virus (SIV) using primers SIV-F1 and SIV-R1 and probe SIV-P1.
FIG. 6 shows the results of real-time PCR (real-time PCR) detection of Porcine Epidemic Diarrhea Virus (PEDV) using primers PEDV-F1 and PEDV-R1 and probe PEDV-P1.
FIG. 7 shows the results of real-time PCR (real-time PCR) detection of porcine circovirus type 2 (PCV2) using primers PCV2-F2 and PCV2-R2 and probe PCV 2-P1.
FIG. 8 shows the results of real-time PCR (real-time PCR) for pseudorabies virus (PRV) using primers PRV-F1 and PRV-R1 and probe PRV-P1.
FIG. 9 shows the results of real-time PCR (real-time PCR) for detecting porcine Severe Valley Virus (SVVA) using primers SVVA-F1 and SVVA-R1 and probe SVVA-P1.
FIG. 10 shows the results of real-time PCR (real-time PCR) for Mycoplasma hyopneumoniae (Mhp) using primers Mhp-F1 and Mhp-R1 and probe Mhp-P1.
Detailed Description
In one aspect, the invention relates to a swine pathogen detection method. In certain embodiments, the method is Polymerase Chain Reaction (PCR). In certain embodiments, the method is reverse transcription-polymerase chain reaction (RT-PCR). In certain embodiments, the method is a thermal convection polymerase chain reaction (cPCR). In certain embodiments, the method is real-time polymerase chain reaction (real-time PCR).
In one aspect, the present invention relates to a swine pathogenic bacteria detection method, comprising:
providing a sample potentially containing one or more nucleotide sequences of porcine pathogens;
providing a pair of oligonucleotide primers defining the 5' ends of two complementary strands of a double stranded target sequence on one or more nucleotide sequences of said pathogenic bacterium;
providing a polymerase;
mixing the sample, the pair of oligonucleotide primers, the polymerase, deoxyadenosine triphosphates (dATPs), deoxycytidine triphosphates (dCTPs), deoxyguanosine triphosphates (dGTPs), and deoxythymidine triphosphates (dTTPs) in a vessel to form a Polymerase Chain Reaction (PCR) mixture;
subjecting the PCR mixture to a thermal convection polymerase chain reaction (cPCR) by heating the bottom of the vessel at a fixed temperature to form a PCR product; and
detecting the PCR product to identify the double-stranded target sequence.
In another aspect, the present invention relates to a swine pathogenic bacteria detection method, comprising:
providing a sample potentially containing one or more nucleotide sequences of porcine pathogens;
providing a pair of oligonucleotide primers defining the 5' ends of two complementary strands of a double stranded target sequence on one or more nucleotide sequences of said pathogenic bacterium;
providing an oligonucleotide probe comprising a sequence complementary to a segment of the double-stranded target sequence, a fluorescent molecule attached to a first location on the oligonucleotide probe, and a fluorescence-suppressing molecule attached to a second location on the oligonucleotide probe, the fluorescence-suppressing molecule substantially suppressing the fluorescent molecule when the oligonucleotide probe is not hybridized to the segment of the double-stranded target sequence and the fluorescent molecule being substantially uninhibited when the oligonucleotide probe is hybridized to the segment of the double-stranded target sequence;
providing a polymerase;
mixing the sample, the pair of oligonucleotide primers, the oligonucleotide probe, the polymerase, deoxyadenosine triphosphates (dATPs), deoxycytidine triphosphates (dCTPs), deoxyguanosine triphosphates (dGTPs), and deoxythymidine triphosphates (dTTPs) in a vessel to form a Polymerase Chain Reaction (PCR) mixture;
subjecting the PCR mixture to a thermal convection polymerase chain reaction (cPCR) by heating the bottom of the vessel at a fixed temperature to form a PCR product; and
detecting the PCR product to identify the double-stranded target sequence.
In yet another aspect, the invention relates to a pair of oligonucleotides for detecting porcine pathogens.
In a further aspect, the invention relates to an oligonucleotide pair and an oligonucleotide probe for detecting porcine pathogens.
It is further understood that in certain embodiments, the oligonucleotide pairs and/or oligonucleotide probes disclosed herein may be used in variations of various basic PCR techniques, such as, but not limited to, reverse transcription polymerase chain reaction (RT-PCR), thermal convection polymerase chain reaction (cPCR), real-time polymerase chain reaction (real-time PCR), nested polymerase chain reaction (nested PCR), and thermal asymmetric multiplex polymerase chain reaction (TAIL-PCR).
As used herein, the term "porcine pathogenic bacteria" means viral, bacterial and parasitic pathogenic bacteria found in and out of swine. Examples of porcine pathogens include, but are not limited to: a) virus: classical Swine Fever Virus (CSFV), Foot and Mouth Disease Virus (FMDV), Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), swine influenza a virus (SIV), Porcine Epidemic Diarrhea Virus (PEDV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine Sendai Valley Virus A (SVVA); b) bacteria: escherichia coli (Escherichia coli), Pasteurella multocida (Pasteurella multocida), Streptococcus suis (Streptococcus suis), Haemophilus parasuis (Haemophilus parauis), Mycoplasma hyorhinis (Mycoplasma hyorhinis), Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), Salmonella choleraesuis (Salmonella choleraesuis), Salmonella saxifragi (Salmonella spp.), Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Clostridium perfringens (Clostridium fricense), Bordetella bronchiseptica (Bordetella bronchus), Campylobacter bacterium (Lawsonia intracellula intracellularis), Salvia suis (Staphylococcus aureus), Rhodococcus erythropolis (Staphylococcus aureus), Staphylococcus aureus (Staphylococcus aureus); c) parasite: sarcoptes suis, trichuris suis, Ascaris suum.
As used herein, the term "thermal convection polymerase chain reaction (cPCR)" refers to a polymerase chain reaction in which the bottom of a tubular container holding a PCR sample is embedded in a stable heat source and the parameters of the PCR, including the total volume, viscosity, surface temperature of the PCR sample, and the inner diameter of the tubular container, are controlled such that the bottom-to-top temperature gradient of the PCR sample is reduced, thermal convection is induced and denaturation, adhesion, polymerization of the PCR sample occur sequentially and repeatedly in different regions of the tubular container. For a detailed description of the thermal convection polymerase chain reaction (cPCR), see, e.g., U.S. patent No. 8,187,813, which is incorporated by reference herein in its entirety.
As used herein, the term "fluorescent molecule" means a substance, or a portion thereof, that is capable of exhibiting fluorescence in a detectable range. As used herein, the term "fluorescence-inhibiting molecule" means a substance or portion thereof that is capable of inhibiting the fluorescence emitted by the fluorescent molecule when excited by a light source. In certain embodiments, the terms "fluorescent molecule" and "fluorescence-inhibiting molecule" are TaqManTMFluorescent molecules and fluorescence-inhibiting molecules of an assay kit (Applied Biosystems inc., california, usa). TaqManTMFor a detailed description of the assay kit see, e.g., Holland et al, Proc. Natl. Acad. Sci, USA (1991)88: 7276-; U.S. Pat. nos. 5,538,848, 5,723,591, 5,876,930, and 7,413,708, all of which are incorporated by reference herein in their entirety.
Examples of such fluorescent molecules include, but are not limited to, 3- (. epsilon. -carboxy) -3'-ethyl-5,5' -dimethylhexacyanine (3- (. epsilon. -carboxypentyl) -3'-ethyl-5,5' -dimethyloxa-carbanine, CYA), 6-carboxyfluorescein (6-carboxyfluorescein, FAM), 5,6-carboxyrhodamine-110 (5,6-carboxyrhodamine-110, R110), 6-carboxyrhodamine-6G (6-carboxyrhodamine-6G, R6G), N ', N' -tetramethyl-6-carboxyrhodamine (N ', N', N ', N' -tetramethylol-6-carboxyrhodamine, TAMRA), 6-carboxy-X-rhodamine (6-carboxyrhodamine-X-rhodomine, ROX), 2',4',5',7' -tetrachloro 4-7-dichlorofluorescein (2',4',5',7' -tetrachlororo-4-7-dichlorofluorescein, TET), 2',7-dimethoxy-4',5'-6carboxyrhodamine (2',7-dimethoxy-4',5' -6 carboxyyredamine, JOE), 6-carboxy-2',4,4',5',7,7' -hexachlorofluorescein (6-carboxy-2',4,4',5',7,7' -hexachlorofluorescein, HEX), ALEXA fluorescence, Cy3 fluorescence and Cy5 fluorescence. Examples of such fluorescence-inhibiting molecules include, but are not limited to, 4- (4'-dimethylamino-phenylazo) benzoic acid (4- (4' -dimethylamino-phenylazo) -benzoic acid, Dabcyl, Black Hole fluorescence inhibitor 1(Black Hole Quencher 1, BHQ1), Black Hole fluorescence inhibitor 2(Black Hole Quencher 2, BHQ2), Black Hole fluorescence inhibitor 3(Black Hole Quencher 3, BHQ3), dihydrocyclo-pyrroloindole tripeptide minor groove conjugate (MGB), tetramethylrhodamine (tetramethylrhodamine, TAMRA). In certain embodiments, the fluorescent molecule is 6-carboxyfluorescein (FAM) and the fluorescence-inhibiting molecule is a dihydrocyclo-pyrroloindole tripeptide Minor Groove Binder (MGB). In certain embodiments, the fluorescent molecule is 6-carboxyfluorescein (FAM) and the fluorescence-inhibiting molecule is black hole fluorescence inhibitor 1(BHQ 1).
Unless defined otherwise herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The methods and techniques of the present invention may generally be performed according to conventional methods known in the art. Generally, the nomenclature used herein to refer to the techniques described below, as well as the biochemical, enzymatic, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization reactions, are those well known in the art and are commonly employed. Unless otherwise indicated, the methods and techniques of the present invention can be generally performed according to conventional methods known in the art and are described in various general and more specific references that are cited and discussed in the present specification.
The invention is further illustrated by the following examples, which should not be construed as further limiting in any way. The entire contents of all cited documents (including references, approved patents, published patent applications, and patent applications filed herewith) cited in this application are hereby expressly incorporated by reference herein.
Examples
Example 1 detection of pathogenic bacteria Classical Swine Fever Virus (CSFV)
A fragment of the polyprotein gene (GenBank access No. AF531433) of the pathogen Classical Swine Fever Virus (CSFV) is inserted into a cloning vector, which may be, but is not limited to, pUC57, pGEM-T, to obtain the pCSFV plasmid.
1. Conventional polymerase chain reaction
A50. mu.l PCR mix used to perform conventional PCR contained: 106Copies of pCSFV plasmid, 0.01-2. mu.M forward primer, 0.01-2. mu.M reverse primer, 0.2. mu.M dNTP and 1.25U Taq DNA polymerase. The amplification reaction was performed in a thermal cycler (e.g., but not limited to PC818, Astec co. ltd., japan) and included an initial cycle of denaturation at 94 ℃ for 3 minutes, and 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds. The amplified product was then analyzed on a 15% polyacrylamide gel in TAE buffer (40mM Tris,20mM acetic acid,1mM EDTA) and visualized by staining with ethidium bromide.
The results of conventional PCR are shown in Table 1, where each primer pair amplified a fragment of the correct size for each target sequence, whereas in the negative control group no target sequence was amplified (results not shown). The results indicate that each primer pair can be used in a conventional PCR amplification reaction to detect the presence of Classical Swine Fever Virus (CSFV).
TABLE 1 results of conventional PCR for each primer pair
Forward primer Reverse primer Synthetic fragment size (bp)
CSFV-F1(SEQ ID NO:1) CSFV-R1(SEQ ID NO:7) 84
CSFV-F2(SEQ ID NO:2) CSFV-R2(SEQ ID NO:8) 71
CSFV-F5(SEQ ID NO:5) CSFV-R2(SEQ ID NO:8) 77
CSFV-F3(SEQ ID NO:3) CSFV-R3(SEQ ID NO:9) 88
CSFV-F4(SEQ ID NO:4) CSFV-R4(SEQ ID NO:10) 76
CSFV-F4(SEQ ID NO:4) CSFV-R5(SEQ ID NO:11) 80
CSFV-F6(SEQ ID NO:6) CSFV-R4(SEQ ID NO:10) 82
CSFV-F6(SEQ ID NO:6) CSFV-R5(SEQ ID NO:11) 86
2. Thermal convection polymerase chain reaction (cPCR)
50 μ l of the PCR mixture contained 5 μ l of RNA of Classical Swine Fever Virus (CSFV) polyprotein gene (10 each)2,103,104,105,106Copy number/. mu.l), 0.01-2. mu.M forward primer, 0.01-2. mu.M reverse primer, 0.01-2. mu.M probe (5 'end and 3' end of each probe sequence are respectively connected with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescence inhibitor molecule black hole fluorescence inhibitor 1(BHQ1)), 1 xcPCR buffer solution, 1-5U reverse transcriptase and 1-5U Taq DNA polymerase. The PCR mixture was added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) apparatus for a specified period of time (about 30-45 minutes). FAM fluorescence in each sample was detected with the cPCR instrument. The above-described cPCR assay was repeated 8 times (n-8) to evaluate the sensitivity of each primer pair to the probe.
The results of the sensitivity test are shown in Table 2, and each combination of the primer pair and the probe can detect 10 in the sample with 100% accuracy2The Classical Swine Fever Virus (CSFV) RNA with the copy number/mu l has the sensitivity of 102Number of copies/. mu.l.
TABLE 2 results of sensitivity test of each primer pair in combination with a probe (n. 8)
Figure BDA0003187955750000211
Figure BDA0003187955750000221
In addition, different pig pathogen gene plasmids (10)6Copy number/. mu.l) as a cPCR template to analyze the specificity of each primer pair and probe combination. The cPCR method was as described above. The results of the specificity test are shown in Table 3, and the combination of each primer pair and probe can correctly detect Classical Swine Fever Virus (CSFV)Samples containing Foot and Mouth Disease Virus (FMDV), Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), swine influenza virus A (SIV), Porcine Epidemic Diarrhea Virus (PEDV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV) could not be detected. The results show that each primer pair has specificity in combination with a probe.
TABLE 3 specificity test results for each primer pair and probe combination
Figure BDA0003187955750000222
Figure BDA0003187955750000231
"+" indicates that a fluorescent signal was detected in the sample, and "-" indicates that no fluorescent signal was detected in the sample.
3. Real-time polymerase chain reaction (real-time PCR, qPCR)
Diluted pCSFV plasmid (10 each)1,102,103,104,105,106,107Number of copies) in a real-time PCR instrument (e.g., without limitation, ABI steponeplus; applied biosystems, Life Technologies, ca, usa) for real-time PCR analysis. Real-time PCR analysis was performed using a commercial RT-PCR Kit (for example, but not limited to, OneStep PrimeScript RT-PCR Kit; Takara Bio Inc., Japan) containing 2. mu.l of pCSFV plasmid, 0.01-2. mu.M of forward primer CSFV-F1(SEQ ID NO:1), 0.01-2. mu.M of reverse primer CSFV-R1(SEQ ID NO:7), and 0.01-2. mu.M of probe CSFV-P1(5'FAM-CCCTGGGTGGTCTAAGTCCTGAGTACAGG-BHQ 13', SEQ ID NO:12) in a total volume of 20. mu.l. The real-time PCR program was 42 ℃ for 5 minutes, 94 ℃ for 10 seconds, and 40 cycles of 94 ℃ for 10 seconds and 60 ℃ for 30 minutes. The results of the fluorescence measurements were recorded during the 60 ℃ procedure.
As shown in fig. 1, a standard curve for real-time PCR analysis of serially diluted (10-fold) pCSFV plasmids was calculated. At least 10 copies of the pCSFV plasmid were detected. R of the standard curve2The value was 0.9965, indicating that the primer pairs and probes of the invention can be used for real-time PCR and produce reliable results.
The results show that the primer pair and the probe of the invention can be used for a cPCR amplification reaction to detect the existence of Classical Swine Fever Virus (CSFV), and have high sensitivity and specificity.
Example 2 detection of pathogenic bacteria Foot and Mouth Disease Virus (FMDV)
A fragment of the polyprotein gene (GenBank access No. MF461724.1) of the pathogenic bacterium foot-and-mouth disease virus (FMDV) is inserted into a cloning vector, which may be, but is not limited to, pUC57, pGEM-T, to obtain pFMDV plasmid.
1. Conventional polymerase chain reaction
A50. mu.l PCR mix used to perform conventional PCR contained: 106Copy number pFMDV plasmid, 0.01-2 μ M forward primer, 0.01-2 μ M reverse primer, 0.2 μ M dNTP and 1.25U Taq DNA polymerase. The amplification reaction was performed in a thermal cycler (e.g., but not limited to PC818, Astec co. ltd., japan) and included an initial cycle of denaturation at 94 ℃ for 3 minutes, and 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds. The amplified product was then analyzed on a 15% polyacrylamide gel in TAE buffer (40mM Tris,20mM acetic acid,1mM EDTA) and visualized by staining with ethidium bromide.
The results of conventional PCR are shown in Table 4, where each primer pair amplified a fragment of the correct size for each target sequence, whereas in the negative control group no target sequence was amplified (results not shown). The results indicate that each primer pair can be used in a conventional PCR amplification reaction to detect the presence of Foot and Mouth Disease Virus (FMDV).
TABLE 4 results of conventional PCR for each primer pair
Forward primer Reverse primer Synthetic fragment size (bp)
FMDV-F1(SEQ ID NO:16) FMDV-R1(SEQ ID NO:22) 88
FMDV-F2(SEQ ID NO:17) FMDV-R2(SEQ ID NO:23) 87
FMDV-F5(SEQ ID NO:20) FMDV-R2(SEQ ID NO:23) 91
FMDV-F3(SEQ ID NO:18) FMDV-R3(SEQ ID NO:24) 91
FMDV-F5(SEQ ID NO:20) FMDV-R3(SEQ ID NO:24) 94
FMDV-F4(SEQ ID NO:19) FMDV-R4(SEQ ID NO:25) 73
FMDV-F4(SEQ ID NO:19) FMDV-R5(SEQ ID NO:26) 81
FMDV-F6(SEQ ID NO:21) FMDV-R4(SEQ ID NO:25) 118
FMDV-F6(SEQ ID NO:21) FMDV-R5(SEQ ID NO:26) 126
2. Thermal convection polymerase chain reaction (cPCR)
50 μ l of the PCR mixture contained 5 μ l of RNA of foot-and-mouth disease Virus (FMDV) polyprotein gene (10 each)2,103,104,105,106Number of copies/. mu.l) or pFMDV plasmid (10 each)2,103,104,105,106Copy number), 0.01-2 μ M forward primer, 0.01-2 μ M reverse primer, 0.01-2 μ M probe (5 'end and 3' end of each probe sequence are respectively connected with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescence inhibitor molecule black hole fluorescence inhibitor 1(BHQ1)), 1 × cPCR buffer solution, 1-5U Taq DNA polymerase and 1-5U reverse transcriptase. The PCR mixture was added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) apparatus for a specified period of time (about 30-45 minutes). FAM fluorescence in each sample was detected with the cPCR instrument. The above-described cPCR assay was repeated 8 times (n-8) to evaluate the sensitivity of each primer pair to the probe.
The results of the sensitivity test are shown in Table 5, and each combination of the primer pair and the probe can detect 10 in the sample with 100% accuracy2Single copy number/. mu.l Foot and Mouth Disease Virus (FMDV) RNA or 102The sensitivity of pFMDV plasmid with single copy number can reach 102Number of copies/. mu.l or 102Number of copies.
TABLE 5 results of sensitivity test of each primer set in combination with a probe (n. 8)
Figure BDA0003187955750000251
Figure BDA0003187955750000261
In addition, different pig pathogen gene plasmids (10)6Number of copies/. mu.l) as a template for cPCR analysis of the specificity of each primer pair in combination with the probe. The cPCR method was as described above. The results of the specificity tests are shown in table 6, and the combination of each primer pair and the probe can correctly detect the sample containing foot-and-mouth disease virus (FMDV), but can not detect the sample containing Classical Swine Fever Virus (CSFV), Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), swine influenza a virus (SIV), Porcine Epidemic Diarrhea Virus (PEDV), porcine circovirus type 2 (PCV2) and pseudorabies virus (PRV). The results show that each primer pair has specificity in combination with a probe.
TABLE 6 specificity test results for each primer pair and Probe combination
Figure BDA0003187955750000262
Figure BDA0003187955750000271
"+" indicates that a fluorescent signal was detected in the sample, and "-" indicates that no fluorescent signal was detected in the sample.
3. Real-time polymerase chain reaction (real-time PCR, qPCR)
Diluted pFMDV plasmid (10 each)1,102,103,104,105,106,107Number of copies) in a real-time PCR instrument (e.g., without limitation, ABI steponeplus; applied biosystems, Life Technologies, ca, usa) for real-time PCR analysis. A commercial RT-P containing 2. mu.l pFMMDV plasmid, 0.01-2. mu.M forward primer FMDV-F1(SEQ ID NO:16), 0.01-2. mu.M reverse primer FMDV-R1(SEQ ID NO:22), 0.01-2. mu.M probe FMDV-P1(5'FAM-CCTTTGCACGCCGTGGGACCA-BHQ 13', SEQ ID NO:27) in a total volume of 20. mu.l was usedCR kits (such as, but not limited to, OneStep PrimeScriptTM RT-PCR Kit; Takara Bio Inc., Japan) for real-time PCR analysis. The real-time PCR program was 42 ℃ for 5 minutes, 94 ℃ for 10 seconds, and 40 cycles of 94 ℃ for 10 seconds and 60 ℃ for 30 minutes. The results of the fluorescence measurements were recorded during the 60 ℃ procedure.
As shown in fig. 2, a standard curve for real-time PCR analysis of serial dilutions (10-fold) of the pFMDV plasmid was calculated. At least 10 copies of the pFMDV plasmid were detected. R of the standard curve2A value of 0.9998 indicates that the primer pairs and probes of the invention can be used for real-time PCR and produce reliable results.
The results indicate that the primer pairs and probes of the invention can be used in a cPCR amplification reaction to detect the presence of Foot and Mouth Disease Virus (FMDV), and have high sensitivity and specificity.
Example 3 detection of pathogenic bacterium Japanese Encephalitis Virus (JEV)
The NS1 gene (GenBank accession No. AB051292) of pathogenic bacterium Japanese Encephalitis Virus (JEV) is inserted into a cloning vector which can be, but is not limited to, pUC57 and pGEM-T to obtain pJEV plasmid.
1. Conventional polymerase chain reaction
A50. mu.l PCR mix used to perform conventional PCR contained: 106Copy number of pJEV plasmid, 0.01-2. mu.M forward primer, 0.01-2. mu.M reverse primer, 0.2. mu.M dNTP and 1.25U Taq DNA polymerase. The amplification reaction was performed in a thermal cycler (e.g., but not limited to PC818, Astec co. ltd., japan) and included an initial cycle of denaturation at 94 ℃ for 3 minutes, and 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds. The amplified product was then analyzed on a 15% polyacrylamide gel in TAE buffer (40mM Tris,20mM acetic acid,1mM EDTA) and visualized by staining with ethidium bromide.
The results of conventional PCR are shown in Table 7, where each primer pair amplified a fragment of the correct size for each target sequence, whereas in the negative control group no target sequence was amplified (results not shown). The results indicate that each primer pair can be used in a conventional PCR amplification reaction to detect the presence of Japanese Encephalitis Virus (JEV).
TABLE 7 results of conventional PCR for each primer pair
Forward primer Reverse primer Synthetic fragment size (bp)
JEV-F1(SEQ ID NO:31) JEV-R1(SEQ ID NO:35) 86
JEV-F1(SEQ ID NO:31) JEV-R2(SEQ ID NO:36) 83
JEV-F2(SEQ ID NO:32) JEV-R1(SEQ ID NO:35) 83
JEV-F3(SEQ ID NO:33) JEV-R1(SEQ ID NO:35) 72
JEV-F3(SEQ ID NO:33) JEV-R2(SEQ ID NO:36) 69
JEV-F4(SEQ ID NO:34) JEV-R1(SEQ ID NO:35) 81
JEV-F4(SEQ ID NO:34) JEV-R2(SEQ ID NO:36) 78
2. Thermal convection polymerase chain reaction (cPCR)
50 μ l of the PCR mixture contained pJEV plasmid (10 each)2,103,104,105,106Copy number), 0.01-2 μ M forward primer, 0.01-2 μ M reverse primer, 0.01-2 μ M probe (5 'end and 3' end of each probe sequence are respectively connected with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescence inhibitor molecule black hole fluorescence inhibitor 1(BHQ1)), 1 xcPCR buffer solution and 1-5U Taq DNA polymerase. The PCR mixture was added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) apparatus for a specified period of time (about 30-45 minutes). FAM fluorescence in each sample was detected with the cPCR instrument. The above-described cPCR assay was repeated 8 times (n-8) to evaluate the sensitivity of each primer pair to the probe.
The results of the sensitivity test are shown in Table 8, and each combination of the primer pair and the probe can detect 10 in the sample with 100% accuracy2The sensitivity of pJEV plasmid with single copy number can reach 102Number of copies.
TABLE 8 results of sensitivity test of each primer set in combination with a probe (n. 8)
Figure BDA0003187955750000291
In addition, different pig pathogen gene plasmids (10)6Number of copies/. mu.l) as a template for cPCR analysis of the specificity of each primer pair in combination with the probe. The cPCR method was as described above. The results of specificity tests are shown in Table 9, and all combinations of primer pairs and probes can correctly detect Japanese Encephalitis Virus (JEV) -containing samples, but cannot detect ancient encephalitis virus (JEV) -containing samplesSamples of Classical Swine Fever Virus (CSFV), Foot and Mouth Disease Virus (FMDV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), swine influenza virus A (SIV), Porcine Epidemic Diarrhea Virus (PEDV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV). The results show that each primer pair has specificity in combination with a probe.
TABLE 9 specificity test results for each primer pair and Probe combination
Figure BDA0003187955750000301
"+" indicates that a fluorescent signal was detected in the sample, and "-" indicates that no fluorescent signal was detected in the sample.
3. Real-time polymerase chain reaction (real-time PCR, qPCR)
Diluted pJEV plasmid (10 each)1,102,103,104,105,106,107Copy number) in a real-time PCR instrument (e.g., without limitation, ABI steponeplus; applied biosystems, Life Technologies, ca, usa) for real-time PCR analysis. Real-time PCR analysis was performed with a commercial RT-PCR Kit (e.g., but not limited to, OnESTep PrimeScript RT-PCR Kit; Takara Bio Inc., Japan) containing 2. mu.l of pJEV plasmid, 0.01-2. mu.M forward primer JEV-F4(SEQ ID NO:34), 0.01-2. mu.M reverse primer JEV-R1(SEQ ID NO:35), 0.01-2. mu.M probe JEV-P1(5'FAM-CCTTCGCAAGAGGTGGACGGCC-BHQ 13', SEQ ID NO:37) in a total volume of 20. mu.l. The real-time PCR program was 42 ℃ for 5 minutes, 94 ℃ for 10 seconds, and 40 cycles of 94 ℃ for 10 seconds and 60 ℃ for 30 minutes. The results of the fluorescence measurements were recorded during the 60 ℃ procedure.
As shown in FIG. 3, a standard curve for real-time PCR analysis of serial dilutions (10-fold) of pJEV plasmid was calculated. At least 10 copies of pJEV plasmid could be detected. R of the standard curve2The value was 0.996, indicating that the primer pairs and probes of the invention can be used for real-time PCR and produce reliable results.
The result shows that the primer pair and the probe can be used for cPCR amplification reaction to detect the existence of Japanese Encephalitis Virus (JEV), and have high sensitivity and specificity.
Example 4 detection of pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)
The ORF6(M matrix protein) gene of the pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) (GenBank accession No. EF536003) was inserted into a cloning vector, which can be, but is not limited to, pUC57, pGEM-T, to obtain pPRRSV plasmid.
1. Conventional polymerase chain reaction
A50. mu.l PCR mix used to perform conventional PCR contained: 106Copy number of pPRRSV plasmid, 0.01-2. mu.M forward primer, 0.01-2. mu.M reverse primer, 0.2. mu.M dNTP and 1.25U Taq DNA polymerase. The amplification reaction was performed in a thermal cycler (e.g., but not limited to PC818, Astec co. ltd., japan) and included an initial cycle of denaturation at 94 ℃ for 3 minutes, and 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds. The amplified product was then analyzed on a 15% polyacrylamide gel in TAE buffer (40mM Tris,20mM acetic acid,1mM EDTA) and visualized by staining with ethidium bromide.
The results of conventional PCR are shown in Table 10, where each primer pair amplified a fragment of the correct size for each target sequence, whereas in the negative control group no target sequence was amplified (results not shown). The results indicate that each primer pair can be used in a conventional PCR amplification reaction to detect the presence of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
TABLE 10 results of conventional PCR for each primer pair
Figure BDA0003187955750000311
Figure BDA0003187955750000321
2. Thermal convection polymerase chain reaction (cPCR)
50 μ l of PCR mixture containing 5 μ l of pigRNA of reproductive and respiratory syndrome Virus (PRRSV) ORF6 gene (10 each)2,103,104,105,106Copy number/. mu.l), 0.01-2. mu.M forward primer, 0.01-2. mu.M reverse primer, 0.01-2. mu.M probe (5 'end and 3' end of each probe sequence are respectively connected with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescence inhibitor molecule black hole fluorescence inhibitor 1(BHQ1)), 1 xcPCR buffer solution, 1-5U reverse transcriptase and 1-5U Taq DNA polymerase. The PCR mixture was added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) apparatus for a specified period of time (about 30-45 minutes). FAM fluorescence in each sample was detected with the cPCR instrument. The above-described cPCR assay was repeated 8 times (n-8) to evaluate the sensitivity of each primer pair to the probe.
The results of the sensitivity test are shown in Table 11, and each combination of the primer pair and the probe can detect 10 in the sample with 100% accuracy2The sensitivity of the RNA of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) with the copy number per mu l can reach 102Number of copies/. mu.l.
TABLE 11 results of sensitivity test of each primer set in combination with a probe (n. 8)
Figure BDA0003187955750000322
Figure BDA0003187955750000331
In addition, different pig pathogen gene plasmids (10)6Number of copies/. mu.l) as a template for cPCR analysis of the specificity of each primer pair in combination with the probe. The cPCR method was as described above. The results of the specificity tests are shown in table 12, and the combination of each primer pair and probe can correctly detect the sample containing Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), but can not detect the sample containing Classical Swine Fever Virus (CSFV), Foot and Mouth Disease Virus (FMDV), encephalitis b virus (JEV), swine influenza a virus (SIV), Porcine Epidemic Diarrhea Virus (PEDV), porcine circovirus type 2 (PCV2) and pseudorabies virus (PRV). The knotThe results show that each primer pair has specificity in combination with the probe.
TABLE 12 specificity test results for each primer pair and Probe combination
Figure BDA0003187955750000332
Figure BDA0003187955750000341
"+" indicates that a fluorescent signal was detected in the sample, and "-" indicates that no fluorescent signal was detected in the sample.
3. Real-time polymerase chain reaction (real-time PCR, qPCR)
Diluted pPRRSV plasmid (10 each)1,102,103,104,105,106,107Number of copies) in a real-time PCR instrument (e.g., without limitation, ABI steponeplus; applied biosystems, Life Technologies, ca, usa) for real-time PCR analysis. Real-time PCR analysis was performed with a commercial RT-PCR Kit (e.g., but not limited to, OneStep PrimeScript RT-PCR Kit; Takara Bio Inc., Japan) containing 2. mu.l of pPRRSV plasmid, 0.01-2. mu.M forward primer PRRSV-F1(SEQ ID NO:40), 0.01-2. mu.M reverse primer PRRSV-R2(SEQ ID NO:45), 0.01-2. mu.M probe PRRSV-P2(5'FAM-AGTGCCGCAGGCTTTCATCCGA-BHQ 13', SEQ ID NO:49) in a total volume of 20. mu.l. The real-time PCR program was 42 ℃ for 5 minutes, 94 ℃ for 10 seconds, and 40 cycles of 94 ℃ for 10 seconds and 60 ℃ for 30 minutes. The results of the fluorescence measurements were recorded during the 60 ℃ procedure.
As shown in fig. 4, a standard curve for real-time PCR analysis of serial dilutions (10-fold) of prrsv plasmid was calculated. At least 10 copies of the pprsv plasmid could be detected. R of the standard curve2A value of 0.9991 indicates that the primer pairs and probes of the invention can be used for real-time PCR and produce reliable results.
The results indicate that the primer pairs and probes of the invention can be used in a cPCR amplification reaction to detect the presence of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and are highly sensitive and specific.
Example 5 detection of pathogenic bacterium swine influenza A Virus (SIV)
The matrix protein gene (GenBank access No. MH234432) of the pathogenic bacterium swine influenza A virus (SIV) was inserted into a cloning vector, which may be, but is not limited to, pUC57, pGEM-T, to obtain the pSIV plasmid.
1. Conventional polymerase chain reaction
A50. mu.l PCR mix used to perform conventional PCR contained: 106Copy number of pSIV plasmid, 0.01-2. mu.M forward primer, 0.01-2. mu.M reverse primer, 0.2. mu.M dNTP and 1.25U Taq DNA polymerase. The amplification reaction was performed in a thermal cycler (e.g., but not limited to PC818, Astec co. ltd., japan) and included an initial cycle of denaturation at 94 ℃ for 3 minutes, and 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds. The amplified product was then analyzed on a 15% polyacrylamide gel in TAE buffer (40mM Tris,20mM acetic acid,1mM EDTA) and visualized by staining with ethidium bromide.
The results of conventional PCR are shown in Table 13, where each primer pair amplified a fragment of the correct size for each target sequence, whereas in the negative control group no target sequence was amplified (results not shown). The results indicate that each primer pair can be used in a conventional PCR amplification reaction to detect the presence of swine influenza a virus (SIV).
TABLE 13 results of conventional PCR for each primer pair
Forward primer Reverse primer Synthetic fragment size (bp)
SIV-F1(SEQ ID NO:51) SIV-R1(SEQ ID NO:56) 83
SIV-F1(SEQ ID NO:51) SIV-R2(SEQ ID NO:57) 83
SIV-F2(SEQ ID NO:52) SIV-R1(SEQ ID NO:56) 88
SIV-F3(SEQ ID NO:53) SIV-R1(SEQ ID NO:56) 116
SIV-F4(SEQ ID NO:54) SIV-R1(SEQ ID NO:56) 77
SIV-F5(SEQ ID NO:55) SIV-R1(SEQ ID NO:56) 82
2. Thermal convection polymerase chain reaction (cPCR)
50 μ l of the PCR mixture contained pSIV plasmid (10 each)2,103,104,105,106Copy number), 0.01-2 μ M forward primer, 0.01-2 μ M reverse primer, 0.01-2 μ M probe (5 'end and 3' end of each probe sequence are respectively connected with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescence inhibitor molecule black hole fluorescence inhibitor 1(BHQ1)), 1 xcPCR buffer solution and 1-5U Taq DNA polymerase. Adding the PCR mixture to a reaction tube, and juxtaposingIn a thermal convection polymerase chain reaction (cPCR) apparatus for a specified period of time (about 30-45 minutes). FAM fluorescence in each sample was detected with the cPCR instrument. The above-described cPCR assay was repeated 8 times (n-8) to evaluate the sensitivity of each primer pair to the probe.
The results of the sensitivity test are shown in Table 14, and each combination of the primer pair and the probe can detect 10 in the sample with 100% accuracy2The sensitivity of pSIV plasmid with single copy number can reach 102Number of copies.
TABLE 14 results of sensitivity test of each primer set in combination with a probe (n. 8)
Figure BDA0003187955750000351
Figure BDA0003187955750000361
In addition, different pig pathogen gene plasmids (10)6Number of copies/. mu.l) as a template for cPCR analysis of the specificity of each primer pair in combination with the probe. The cPCR method was as described above. The results of the specificity tests are shown in table 15, and the combination of each primer pair and probe can correctly detect the sample containing swine influenza a virus (SIV), but can not detect the sample containing Classical Swine Fever Virus (CSFV), Foot and Mouth Disease Virus (FMDV), Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Epidemic Diarrhea Virus (PEDV), porcine circovirus type 2 (PCV2) and pseudorabies virus (PRV). The results show that each primer pair has specificity in combination with a probe.
TABLE 15 specificity test results for each primer pair and Probe combination
Figure BDA0003187955750000362
Figure BDA0003187955750000371
"+" indicates that a fluorescent signal was detected in the sample, and "-" indicates that no fluorescent signal was detected in the sample.
3. Real-time polymerase chain reaction (real-time PCR, qPCR)
In diluted pSIV plasmids (10 each)1,102,103,104,105,106,107Copy number) in a real-time PCR instrument (e.g., without limitation, ABI steponeplus; applied biosystems, Life Technologies, ca, usa) for real-time PCR analysis. Real-time PCR analysis was performed using a commercial RT-PCR Kit (e.g., but not limited to, OnESTep PrimeScript RT-PCR Kit; Takara Bio Inc., Japan) containing 2. mu.l of pSIV plasmid, 0.01-2. mu.M of forward primer SIV-F1(SEQ ID NO:51), 0.01-2. mu.M of reverse primer SIV-R1(SEQ ID NO:56), 0.01-2. mu.M of probe SIV-P1(5'FAM-TGAGCGAGGACTGCAGCGTAGACG-BHQ 13', SEQ ID NO:58) in a total volume of 20. mu.l. The real-time PCR program was 42 ℃ for 5 minutes, 94 ℃ for 10 seconds, and 40 cycles of 94 ℃ for 10 seconds and 60 ℃ for 30 minutes. The results of the fluorescence measurements were recorded during the 60 ℃ procedure.
As shown in FIG. 5, a standard curve for real-time PCR analysis of serially diluted (10-fold) pSIV plasmid was calculated. At least 10 copies of the pSIV plasmid could be detected. R of the standard curve2The value was 0.9997, indicating that the primer pairs and probes of the invention can be used for real-time PCR and produce reliable results.
The results indicate that the primer pairs and probes of the invention can be used in a cPCR amplification reaction to detect the presence of swine influenza A virus (SIV) and are highly sensitive and specific.
Example 6 detection of the pathogenic bacterium Porcine Epidemic Diarrhea Virus (PEDV)
Respectively inserting 5 'untranslated region (5' -UTR), nucleocapsid protein gene (nucleocapsid protein) and spinous process protein gene (spike protein) in genome sequence (GenBank access No. AF353511) of pathogenic Porcine Epidemic Diarrhea Virus (PEDV) into a cloning vector, wherein the cloning vector can be, but is not limited to, pUC57 and pGEM-T, so as to respectively obtain pPEDV-5UTR, pPEDV-NC and pPEDV-S plasmids.
1. Conventional polymerase chain reaction
A50. mu.l PCR mix used to perform conventional PCR contained: 106Plasmids of several copies (pPEDV-5 UTR, pPEDV-NC, pPEDV-S plasmids, respectively), 0.01-2. mu.M forward primer, 0.01-2. mu.M reverse primer, 0.2. mu.M dNTP and 1.25U Taq DNA polymerase. The amplification reaction was performed in a thermal cycler (e.g., but not limited to PC818, Astec co. ltd., japan) and included an initial cycle of denaturation at 94 ℃ for 3 minutes, and 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds. The amplified product was then analyzed on a 15% polyacrylamide gel in TAE buffer (40mM Tris,20mM acetic acid,1mM EDTA) and visualized by staining with ethidium bromide.
The results of conventional PCR are shown in Table 16, where each primer pair amplified a fragment of the correct size for each target sequence, whereas in the negative control group no target sequence was amplified (results not shown). The results indicate that each primer pair can be used in a conventional PCR amplification reaction to detect the presence of Porcine Epidemic Diarrhea Virus (PEDV).
TABLE 16 results of conventional PCR for each primer pair
Forward primer Reverse primer Synthetic fragment size (bp)
PEDV-F1(SEQ ID NO:61) PEDV-R1(SEQ ID NO:67) 97
PEDV-F2(SEQ ID NO:62) PEDV-R2(SEQ ID NO:68) 67
PEDV-F3(SEQ ID NO:63) PEDV-R3(SEQ ID NO:69) 90
PEDV-F3(SEQ ID NO:63) PEDV-R5(SEQ ID NO:71) 84
PEDV-F4(SEQ ID NO:64) PEDV-R4(SEQ ID NO:70) 72
PEDV-F5(SEQ ID NO:65) PEDV-R2(SEQ ID NO:68) 70
PEDV-F6(SEQ ID NO:66) PEDV-R4(SEQ ID NO:70) 93
PEDV-F6(SEQ ID NO:66) PEDV-R6(SEQ ID NO:72) 88
2. Thermal convection polymerase chain reaction (cPCR)
50 μ l of the PCR mixture contained 5 μ l of 5 'untranslated region (5' -UTR) of Porcine Epidemic Diarrhea Virus (PEDV), nucleocapsid protein gene, RNA of spinous Process protein gene (10 each)2,103,104,105,106Number of copies/. mu.l), 001-2 μ M forward primer, 0.01-2 μ M reverse primer, 0.01-2 μ M probe (5 'end and 3' end of each probe sequence are respectively connected with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescence inhibitor molecule black hole fluorescence inhibitor 1(BHQ1)), 1 xcPCR buffer solution, 1-5U reverse transcriptase and 1-5U Taq DNA polymerase. The PCR mixture was added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) apparatus for a specified period of time (about 30-45 minutes). FAM fluorescence in each sample was detected with the cPCR instrument. The above-described cPCR assay was repeated 8 times (n-8) to evaluate the sensitivity of each primer pair to the probe.
The results of the sensitivity test are shown in Table 17, and each combination of the primer pair and the probe can detect 10 in the sample with 100% accuracy2The sensitivity of the Porcine Epidemic Diarrhea Virus (PEDV) with the copy number per mu l can reach 102Number of copies/. mu.l.
TABLE 17 results of sensitivity test of each primer set in combination with a probe (n. 8)
Figure BDA0003187955750000391
Figure BDA0003187955750000401
In addition, different pig pathogen gene plasmids (10)6Number of copies/. mu.l) as a template for cPCR analysis of the specificity of each primer pair in combination with the probe. The cPCR method was as described above. The results of the specificity tests are shown in table 18, and the combination of each primer pair and probe can correctly detect the sample containing the Porcine Epidemic Diarrhea Virus (PEDV), but can not detect the sample containing Classical Swine Fever Virus (CSFV), Foot and Mouth Disease Virus (FMDV), Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), swine influenza a virus (SIV), porcine circovirus type 2 (PCV2) and pseudorabies virus (PRV). The results show that each primer pair has specificity in combination with a probe.
TABLE 18 specificity test results for each primer pair and Probe combination
Figure BDA0003187955750000402
Figure BDA0003187955750000411
"+" indicates that a fluorescent signal was detected in the sample, and "-" indicates that no fluorescent signal was detected in the sample.
3. Real-time polymerase chain reaction (real-time PCR, qPCR)
Diluted pPEDV-5UTR plasmid (10 each)1,102,103,104,105,106,107Number of copies) in a real-time PCR instrument (e.g., without limitation, ABI steponeplus; applied biosystems, Life Technologies, ca, usa) for real-time PCR analysis. Real-time PCR analysis was performed using a commercial RT-PCR Kit (e.g., but not limited to, OneStep PrimeScript RT-PCR Kit; Takara Bio Inc., Japan) containing 2. mu.l of pPEDV-5UTR plasmid, 0.01-2. mu.M of forward primer PEDV-F1(SEQ ID NO:61), 0.01-2. mu.M of reverse primer PEDV-R1(SEQ ID NO:67), 0.01-2. mu.M of probe PEDV-P1(5'FAM-TGTCCTCTAGTTCCTGGTTGGCGTTCC-BHQ 13', SEQ ID NO:73) in a total volume of 20. mu.l. The real-time PCR program was 42 ℃ for 5 minutes, 94 ℃ for 10 seconds, and 40 cycles of 94 ℃ for 10 seconds and 60 ℃ for 30 minutes. The results of the fluorescence measurements were recorded during the 60 ℃ procedure.
As shown in FIG. 6, a standard curve for real-time PCR analysis of serial dilutions (10 fold) of pPEDV-5UTR plasmid was calculated. At least 10 copies of the pPEDV-5UTR plasmid were detected. R of the standard curve2The value was 0.9974, indicating that the primer pairs and probes of the invention can be used for real-time PCR and produce reliable results.
The results indicate that the primer pairs and probes of the invention can be used in a cPCR amplification reaction to detect the presence of Porcine Epidemic Diarrhea Virus (PEDV), and are highly sensitive and specific.
Example 7 detection of the pathogen porcine circovirus type 2 (PCV2)
The genomic sequence (GenBank accession No. KM487709) of the pathogen porcine circovirus type 2 (PCV2) was inserted into a cloning vector, which can be, but is not limited to, pUC57, pGEM-T, to obtain pPCV2 plasmid.
1. Conventional polymerase chain reaction
A50. mu.l PCR mix used to perform conventional PCR contained: 106pPCV2 plasmid of several copies, 0.01-2. mu.M forward primer, 0.01-2. mu.M reverse primer, 0.2. mu.M dNTP and 1.25U Taq DNA polymerase. The amplification reaction was performed in a thermal cycler (e.g., but not limited to PC818, Astec co. ltd., japan) and included an initial cycle of denaturation at 94 ℃ for 3 minutes, and 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds. The amplified product was then analyzed on a 15% polyacrylamide gel in TAE buffer (40mM Tris,20mM acetic acid,1mM EDTA) and visualized by staining with ethidium bromide.
The results of conventional PCR are shown in Table 19, where each primer pair amplified a fragment of the correct size for each target sequence, whereas in the negative control group no target sequence was amplified (results not shown). The results indicate that each primer pair can be used in a conventional PCR amplification reaction to detect the presence of porcine circovirus type 2 (PCV 2).
TABLE 19 results of conventional PCR for each primer pair
Figure BDA0003187955750000421
2. Thermal convection polymerase chain reaction (cPCR)
50 μ l of the PCR mixture contained pPCV2 plasmid (10 each)2,103,104,105,106Copy number), 0.01-2 μ M forward primer, 0.01-2 μ M reverse primer, 0.01-2 μ M probe (5 'end and 3' end of each probe sequence are respectively connected with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescence inhibitor molecule black hole fluorescence inhibitor 1(BHQ1) or dihydrocyclo-pyrroloindole tripeptide Minor Groove Binder (MGB)), 1 xcPCR buffer solution and 1-5U Taq DNA polymerase. Will PThe CR mix was added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) apparatus for a specified period of time (about 30-45 minutes). FAM fluorescence in each sample was detected with the cPCR instrument. The above-described cPCR assay was repeated 8 times (n-8) to evaluate the sensitivity of each primer pair to the probe.
The results of the sensitivity test are shown in Table 20, and each combination of the primer pair and the probe can detect 10 in the sample with 100% accuracy2The sensitivity of the pPCV2 plasmid with the copy number can reach 102Number of copies.
TABLE 20 results of sensitivity test of each primer set in combination with a probe (n. 8)
Figure BDA0003187955750000431
In addition, different pig pathogen gene plasmids (10)6Number of copies/. mu.l) as a template for cPCR analysis of the specificity of each primer pair in combination with the probe. The cPCR method was as described above. The results of the specificity tests are shown in table 21, and the combination of each primer pair and probe can correctly detect the sample containing porcine circovirus type 2 (PCV2), but can not detect the sample containing Classical Swine Fever Virus (CSFV), Foot and Mouth Disease Virus (FMDV), Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), swine influenza a virus (SIV), Porcine Epidemic Diarrhea Virus (PEDV), pseudorabies virus (PRV). The results show that each primer pair has specificity in combination with a probe.
TABLE 21 specificity test results for each primer pair and Probe combination
Figure BDA0003187955750000441
"+" indicates that a fluorescent signal was detected in the sample, and "-" indicates that no fluorescent signal was detected in the sample.
3. Real-time polymerase chain reaction (real-time PCR, qPCR)
Diluted pPCV2 plasmid (10 each)1,102,103,104,105,106,107Number of copies) in a real-time PCR instrument (e.g., without limitation, ABI steponeplus; applied biosystems, Life Technologies, ca, usa) for real-time PCR analysis. Real-time PCR analysis was performed using a commercial RT-PCR Kit (e.g., but not limited to, OnTep PrimeScript RT-PCR Kit; Takara Bio Inc., Japan) containing 2. mu.l of pPCV2 plasmid, 0.01-2. mu.M forward primer PCV2-F2(SEQ ID NO:78), 0.01-2. mu.M reverse primer PCV2-R2(SEQ ID NO:83), 0.01-2. mu.M probe PCV2-P1(5'FAM-AAGCGGACCCCAACC-MGB 3', SEQ ID NO:86) in a total volume of 20. mu.l. The real-time PCR program was 42 ℃ for 5 minutes, 94 ℃ for 10 seconds, and 40 cycles of 94 ℃ for 10 seconds and 60 ℃ for 30 minutes. The results of the fluorescence measurements were recorded during the 60 ℃ procedure.
As shown in fig. 7, a standard curve for real-time PCR analysis of serial dilutions (10-fold) of the pPCV2 plasmid was calculated. At least 10 copies of the pPCV2 plasmid could be detected. R of the standard curve2A value of 0.9996 indicates that the primer pairs and probes of the invention can be used for real-time PCR and produce reliable results.
The results indicate that the primer pairs and probes of the invention can be used in a cPCR amplification reaction to detect the presence of porcine circovirus type 2 (PCV2), and are highly sensitive and specific.
Example 8 detection of the pathogenic bacterium Pseudorabies Virus (PRV)
The glycoprotein B (gB) gene of the pathogenic pseudorabies virus (PRV) (GenBank accession No. JF797217.1) was inserted into a cloning vector, which may be, but is not limited to, pUC57, pGEM-T, to obtain the pPRV plasmid.
1. Conventional polymerase chain reaction
A50. mu.l PCR mix used to perform conventional PCR contained: 106Copy number of pPRV plasmid, 0.01-2. mu.M forward primer, 0.01-2. mu.M reverse primer, 0.2. mu.M dNTP and 1.25U Taq DNA polymerase. The amplification reaction is performed in a thermal cycler (e.g., but not limited to PC818, Astec co. ltd., japan) and comprises an initial cycle of denaturation at 94 ℃ for 3 minutes, and 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 60 ℃ for 30 secondsAnd 72 ℃ extension for 30 seconds. The amplified product was then analyzed on a 15% polyacrylamide gel in TAE buffer (40mM Tris,20mM acetic acid,1mM EDTA) and visualized by staining with ethidium bromide.
The results of conventional PCR are shown in Table 22, where each primer pair amplified a fragment of the correct size for each target sequence, whereas in the negative control group no target sequence was amplified (results not shown). The results indicate that each primer pair can be used in a conventional PCR amplification reaction to detect the presence of pseudorabies virus (PRV).
TABLE 22 results of conventional PCR for each primer pair
Forward primer Reverse primer Synthetic fragment size (bp)
PRV-F1(SEQ ID NO:89) PRV-R1(SEQ ID NO:96) 62
PRV-F2(SEQ ID NO:90) PRV-R2(SEQ ID NO:97) 95
PRV-F3(SEQ ID NO:91) PRV-R3(SEQ ID NO:98) 120
PRV-F4(SEQ ID NO:92) PRV-R4(SEQ ID NO:99) 60
PRV-F5(SEQ ID NO:93) PRV-R5(SEQ ID NO:100) 87
PRV-F6(SEQ ID NO:94) PRV-R6(SEQ ID NO:101) 71
PRV-F7(SEQ ID NO:95) PRV-R7(SEQ ID NO:102) 61
2. Thermal convection polymerase chain reaction (cPCR)
50 μ l of the PCR mixture contained the pPRV plasmid (10 each)2,103,104,105,106Copy number), 0.01-2 μ M forward primer, 0.01-2 μ M reverse primer, 0.01-2 μ M probe (5 'end and 3' end of each probe sequence are respectively connected with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescence inhibitor molecule black hole fluorescence inhibitor 1(BHQ1)), 1 xcPCR buffer solution and 1-5U Taq DNA polymerase. The PCR mixture was added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) apparatus for a specified period of time (about 30-45 minutes). FAM fluorescence in each sample was detected with the cPCR instrument. The above-described cPCR assay was repeated 8 times (n-8) to evaluate the sensitivity of each primer pair to the probe.
The results of the sensitivity test are shown in Table 23, and each combination of the primer pair and the probe can detect 10 in the sample with 100% accuracy2The sensitivity of the pPRV plasmid with the copy number can reach 102Number of copies.
TABLE 23 results of sensitivity test of each primer set in combination with a probe (n ═ 8)
Figure BDA0003187955750000461
In addition, different pig pathogen gene plasmids (10)6Number of copies/. mu.l) as a template for cPCR analysis of the specificity of each primer pair in combination with the probe. The cPCR method was as described above. The results of the specificity tests are shown in table 24, and the combination of each primer pair and probe can correctly detect the sample containing pseudorabies virus (PRV), but can not detect the sample containing Classical Swine Fever Virus (CSFV), Foot and Mouth Disease Virus (FMDV), Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), swine influenza virus a (SIV), Porcine Epidemic Diarrhea Virus (PEDV), and porcine circovirus type 2 (PCV 2). The results show that each primer pair has specificity in combination with a probe.
TABLE 24 specificity test results for each primer pair and probe combination
Figure BDA0003187955750000471
"+" indicates that a fluorescent signal was detected in the sample, and "-" indicates that no fluorescent signal was detected in the sample.
3. Real-time polymerase chain reaction (real-time PCR, qPCR)
Diluted pPCV2 plasmid (10 each)1,102,103,104,105,106,107Number of copies) in a real-time PCR instrument (e.g., without limitation, ABI steponeplus; applied biosystems, Life Technologies, ca, usa) for real-time PCR analysis. Real-time PCR analysis was performed with a commercial RT-PCR Kit (e.g., but not limited to, OneStep PrimeScript RT-PCR Kit; Takara Bio Inc., Japan) containing 2. mu.l of pPCV2 plasmid, 0.01-2. mu.M forward primer PCV2-F1(SEQ ID NO:89), 0.01-2. mu.M reverse primer PCV2-R1(SEQ ID NO:96), 0.01-2. mu.M probe PCV2-P1(5'FAM-CGGGCTTCTACCACACGGGCA-BHQ 13', SEQ ID NO:103) in a total volume of 20. mu.l. The real-time PCR program was 42 ℃ for 5 minutes, 94 ℃ for 10 seconds, and 40 cycles of 94 ℃ for 10 seconds and 60 ℃ for 30 minutesA clock. The results of the fluorescence measurements were recorded during the 60 ℃ procedure.
As shown in fig. 8, a standard curve for real-time PCR analysis of serial dilutions (10-fold) of the pPCV2 plasmid was calculated. At least 100 copies of the pPCV2 plasmid could be detected. R of the standard curve2A value of 0.9991 indicates that the primer pairs and probes of the invention can be used for real-time PCR and produce reliable results.
The results indicate that the primer pairs and probes of the invention can be used in a cPCR amplification reaction to detect the presence of pseudorabies virus (PRV) and are highly sensitive and specific.
Example 9 detection of the pathogenic Swine Saigae Canavalia Virus (SVVA)
The 3D polymerase (3D ploymerase) gene (GenBank accession No. DQ641257.1) of the pathogenic porcine Selaginella Virus (SVVA) was inserted into a cloning vector, which can be, but is not limited to, pUC57, pGEM-T, to obtain the pSVVA plasmid.
1. Conventional polymerase chain reaction
A50. mu.l PCR mix used to perform conventional PCR contained: 106Copy number of pSVVA plasmid, 0.01-2. mu.M forward primer, 0.01-2. mu.M reverse primer, 0.2. mu.M dNTP and 1.25U Taq DNA polymerase. The amplification reaction was performed in a thermal cycler (e.g., but not limited to PC818, Astec co. ltd., japan) and included an initial cycle of denaturation at 94 ℃ for 3 minutes, and 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds. The amplified product was then analyzed on a 15% polyacrylamide gel in TAE buffer (40mM Tris,20mM acetic acid,1mM EDTA) and visualized by staining with ethidium bromide.
The results of conventional PCR are shown in Table 25, where each primer pair amplified a fragment of the correct size for each target sequence, whereas in the negative control group no target sequence was amplified (results not shown). The results indicate that each primer pair can be used in a conventional PCR amplification reaction to detect the presence of porcine epilaked valley virus (SVVA).
TABLE 25 results of conventional PCR for each primer pair
Figure BDA0003187955750000481
Figure BDA0003187955750000491
2. Thermal convection polymerase chain reaction (cPCR)
50 μ l of the PCR mixture contained 5 μ l of RNA of the porcine Sambucus plus Valley Virus (SVVA)3D polymerase gene (10 each)2,103,104,105,106Number of copies/. mu.l) or pSVVA plasmid (10 each)2,103,104,105,106Copy number), 0.01-2 μ M forward primer, 0.01-2 μ M reverse primer, 0.01-2 μ M probe (5 'end and 3' end of each probe sequence are respectively connected with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescence inhibition molecule dihydrocyclo-pyrroloindole tripeptide Minor Groove Binder (MGB)), 1 xcPCR buffer solution, 1-5U reverse transcriptase and 1-5U Taq DNA polymerase. The PCR mixture was added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) apparatus for a specified period of time (about 30-45 minutes). FAM fluorescence in each sample was detected with the cPCR instrument. The above-described cPCR assay was repeated 8 times (n-8) to evaluate the sensitivity of each primer pair to the probe.
The results of the sensitivity test are shown in Table 26, and each combination of primer pair and probe can detect 10 in the sample with 100% accuracy2Copy number/. mu.l porcine Seneca Valley Virus (SVVA) RNA or 102The sensitivity of pSVVA plasmid with single copy number can reach 102Number of copies/. mu.l or 102Number of copies.
TABLE 26 results of sensitivity test of each primer set in combination with a probe (n. 8)
Figure BDA0003187955750000492
Figure BDA0003187955750000501
In addition, specificity of each primer pair and probe combination was analyzed using different pig pathogen gene plasmids (106 copies/. mu.l) as cPCR templates. The cPCR method was as described above. The results of the specificity tests are shown in table 27, and the combination of each primer pair and probe can correctly detect the sample containing porcine Selaginella virus (SVVA), but can not detect the sample containing Classical Swine Fever Virus (CSFV), Foot and Mouth Disease Virus (FMDV), Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), swine influenza A virus (SIV), Porcine Epidemic Diarrhea Virus (PEDV) and porcine circovirus type 2 (PCV 2). The results show that each primer pair has specificity in combination with a probe.
TABLE 27 specificity test results for each primer pair and Probe combination
Figure BDA0003187955750000502
Figure BDA0003187955750000511
"+" indicates that a fluorescent signal was detected in the sample, and "-" indicates that no fluorescent signal was detected in the sample.
3. Real-time polymerase chain reaction (real-time PCR, qPCR)
Diluted pSVVA plasmid (10 each)1,102,103,104,105,106Number of copies) in a real-time PCR instrument (e.g., without limitation, ABI steponeplus; applied biosystems, Life Technologies, ca, usa) for real-time PCR analysis. A commercial RT-PCR Kit (e.g., but not limited to, OneStep PrimeScript RT-PCR Kit; Takara Bio Inc., Japan) containing 2. mu.l of pSVVA plasmid, 0.01-2. mu.M forward primer SVVA-F1(SEQ ID NO:107), 0.01-2. mu.M reverse primer SVVA-R1(SEQ ID NO:112), 0.01-2. mu.M probe SVVA-P1(5'FAM-AAACCAGGAACACTACTC-MGB 3', SEQ ID NO:117) in a total volume of 20. mu.l was used for real-time PCRAnd (4) PCR analysis. The real-time PCR program was 42 ℃ for 5 minutes, 94 ℃ for 10 seconds, and 40 cycles of 94 ℃ for 10 seconds and 60 ℃ for 30 minutes. The results of the fluorescence measurements were recorded during the 60 ℃ procedure.
As shown in fig. 9, a standard curve for real-time PCR analysis of serially diluted (10-fold) pSVVA plasmids was calculated. At least 100 copies of pSVVA plasmid could be detected. R of the standard curve2A value of 0.9911 indicates that the primer pairs and probes of the invention can be used for real-time PCR and produce reliable results.
The results indicate that the primer pairs and probes of the invention can be used in a cPCR amplification reaction to detect the presence of porcine epididymis valley virus (SVVA), and have high sensitivity and specificity.
Example 10 detection of the pathogen Mycoplasma hyopneumoniae (Mhp)
The P97 gene (GenBank accession No. AE017243.1) of the pathogen Mycoplasma hyopneumoniae (Mhp) was inserted into a cloning vector, which may be, but is not limited to, pUC57, pGEM-T, to obtain the pMhp plasmid.
1. Conventional polymerase chain reaction
A50. mu.l PCR mix used to perform conventional PCR contained: 106Several copies of pMhp plasmid, 0.01-2. mu.M forward primer, 0.01-2. mu.M reverse primer, 0.2. mu.M dNTP and 1.25U Taq DNA polymerase. The amplification reaction was performed in a thermal cycler (e.g., but not limited to PC818, Astec co. ltd., japan) and included an initial cycle of denaturation at 94 ℃ for 3 minutes, and 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds. The amplified product was then analyzed on a 15% polyacrylamide gel in TAE buffer (40mM Tris,20mM acetic acid,1mM EDTA) and visualized by staining with ethidium bromide.
The results of conventional PCR are shown in Table 28, where each primer pair amplified a fragment of the correct size for each target sequence, whereas in the negative control group no target sequence was amplified (results not shown). The results indicate that each primer pair can be used in a conventional PCR amplification reaction to detect the presence of mycoplasma hyopneumoniae (Mhp).
TABLE 28 results of conventional PCR for each primer pair
Forward primer Reverse primer Synthetic fragment size (bp)
Mhp-F1(SEQ ID NO:121) Mhp-R1(SEQ ID NO:126) 82
Mhp-F1(SEQ ID NO:121) Mhp-R5(SEQ ID NO:130) 82
Mhp-F2(SEQ ID NO:122) Mhp-R2(SEQ ID NO:127) 78
Mhp-F3(SEQ ID NO:123) Mhp-R3(SEQ ID NO:128) 68
Mhp-F4(SEQ ID NO:124) Mhp-R4(SEQ ID NO:129) 77
Mhp-F5(SEQ ID NO:125) Mhp-R1(SEQ ID NO:126) 85
Mhp-F5(SEQ ID NO:125) Mhp-R5(SEQ ID NO:130) 85
2. Thermal convection polymerase chain reaction (cPCR)
50 μ l of the PCR mixture contained the RNA of Mycoplasma hyopneumoniae (Mhp) P97 gene (10 each)2,103,104,105,106Number of copies/. mu.l) or pMhp plasmid (10 each)2,103,104,105,106Copy number), 0.01-2 μ M forward primer, 0.01-2 μ M reverse primer, 0.01-2 μ M probe (5 'end and 3' end of each probe sequence are respectively connected with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescence inhibition molecule dihydrocyclo-pyrroloindole tripeptide Minor Groove Binder (MGB)), 1 xcPCR buffer solution, 1-5U reverse transcriptase and 1-5U Taq DNA polymerase. The PCR mixture was added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) apparatus for a specified period of time (about 30-45 minutes). FAM fluorescence in each sample was detected with the cPCR instrument. The above-described cPCR assay was repeated 8 times (n-8) to evaluate the sensitivity of each primer pair to the probe.
The results of the sensitivity test are shown in Table 29, and each combination of the primer pair and the probe can detect 10 in the sample with 100% accuracy2Copies/. mu.l Mycoplasma hyopneumoniae (Mhp) RNA or 102The sensitivity of pMhp plasmid with single copy number can reach 102Number of copies/. mu.l or 102Number of copies.
TABLE 29 results of sensitivity test of each primer set in combination with a probe (n ═ 8)
Figure BDA0003187955750000531
In addition, different pig pathogen gene plasmids (10)6Copy number/. mu.l) as cPCR template for analyzing each of the above primer pairs and probesSpecificity of the combination. The cPCR method was as described above. The results of the specificity tests are shown in table 30, and the combination of each primer pair and probe can correctly detect the sample containing mycoplasma hyopneumoniae (Mhp), but can not detect the sample containing Classical Swine Fever Virus (CSFV), Foot and Mouth Disease Virus (FMDV), Japanese Encephalitis Virus (JEV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), swine influenza virus a (SIV), Porcine Epidemic Diarrhea Virus (PEDV), and porcine circovirus type 2 (PCV 2). The results show that each primer pair has specificity in combination with a probe.
TABLE 30 specificity test results for each primer pair and Probe combination
Figure BDA0003187955750000541
"+" indicates that a fluorescent signal was detected in the sample, and "-" indicates that no fluorescent signal was detected in the sample.
3. Real-time polymerase chain reaction (real-time PCR, qPCR)
In diluted pMhp plasmid (10 each)1,102,103,104,105,106Number of copies) in a real-time PCR instrument (e.g., without limitation, ABI steponeplus; applied biosystems, Life Technologies, ca, usa) for real-time PCR analysis. Real-time PCR analysis was performed using a commercial RT-PCR Kit (e.g., but not limited to, OnStep PrimeScript RT-PCR Kit; Takara Bio Inc., Japan) containing 2. mu.l of pMhp plasmid, 0.01-2. mu.M forward primer Mhp-F1(SEQ ID NO:121), 0.01-2. mu.M reverse primer Mhp-R1(SEQ ID NO:126), 0.01-2. mu.M probe Mhp-P1(5'FAM-AAAGTGCCCGTGAAAT-MGB 3', SEQ ID NO:131) in a total volume of 20. mu.l. The real-time PCR program was 42 ℃ for 5 minutes, 94 ℃ for 10 seconds, and 40 cycles of 94 ℃ for 10 seconds and 60 ℃ for 30 minutes. The results of the fluorescence measurements were recorded during the 60 ℃ procedure.
As shown in FIG. 10, a standard curve for real-time PCR analysis of serially diluted (10-fold) pMhp plasmid was calculated. At least 100 copies of the pMhp plasmid could be detected. R of the standard curve2The value is 0.9985, indicating a primer set of the inventionProbes can be used for real-time PCR and produce reliable results.
The results show that the primer pair and the probe of the invention can be used for a cPCR amplification reaction to detect the existence of mycoplasma hyopneumoniae (Mhp), and have high sensitivity and specificity.
The detailed description is specific to one possible embodiment of the invention, but the embodiment is not intended to limit the scope of the invention, and equivalents and modifications not departing from the technical spirit of the invention are intended to be included in the scope of the invention.
Sequence listing
<110> advanced application technology, Inc
<120> pig pathogenic bacteria detection method
<130> TPF21-01-6290
<150> US63/061,440
<151> 2020-08-05
<160> 134
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gggactagcc gtagtggcg 19
<210> 2
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcgcagtccc cgtacgt 17
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cctgggtggt ctaagtcctg agt 23
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gcaaaacgga gggactagcc a 21
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gtaacatcgc agtccccgta 20
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggactagcaa aacggaggga ct 22
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gcttctgctc acgtcgaact a 21
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
acatccaagt ccgggagagt 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ccctcgtcca cgtagcatct 20
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ctactgacga ctgtcctgta ctc 23
<210> 11
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cgaactactg acgactgtcc tgtac 25
<210> 12
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ccctgggtgg tctaagtcct gagtacagg 29
<210> 13
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gtggtggcaa ccaacgcgat agaatc 26
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
agttcgacgt gagcagaagc ccacc 25
<210> 15
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cgagctccct gggtggtcta agtcct 26
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
aacctgtgat ggcttcgaag ac 22
<210> 17
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
aacctgtgat ggcttcgaag a 21
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
aaacctgtga tggcttcgaa ga 22
<210> 19
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tccactctgg acctgatgag ta 22
<210> 20
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tataaacctg tgatggcttc gaa 23
<210> 21
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gtgggaccat acaggagaag ttg 23
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
cctgccacgg agatcaactt 20
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ctgccacgga gatcaacttc tc 22
<210> 24
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gtcctgccac ggagatcaa 19
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ctgtagcttg gaatctcaaa ga 22
<210> 26
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aaagtgatct gtagcttgga atctca 26
<210> 27
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
cctttgcacg ccgtgggacc a 21
<210> 28
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tctcgaagct atcctctcct ttgcacgc 28
<210> 29
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tgcacgccgt gggaccatac a 21
<210> 30
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cggcgtctct tcgagccctt tcag 24
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tgggccttct ggtgatgttt 20
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gccttctggt gatgtttctg g 21
<210> 33
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
atgtttctgg ccacccagg 19
<210> 34
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
cttctggtga tgtttctggc c 21
<210> 35
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
ccccaaaacc gcaggaa 17
<210> 36
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
caaaaccgca ggaatcgtca at 22
<210> 37
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ccttcgcaag aggtggacgg cc 22
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gtccttcgca agaggtggac ggc 23
<210> 39
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
cttcgcaaga ggtggacggc ca 22
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tgctaggccg caagtacatt 20
<210> 41
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
tctggcccct gcccaccacg tt 22
<210> 42
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gaaattcatc acctccagat g 21
<210> 43
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
cttgctaggc cgcaagtaca t 21
<210> 44
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tgaaagcctg cggcactt 18
<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
cgacaaatgc gtggttatca 20
<210> 46
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
actttcaacg tggtgggcag g 21
<210> 47
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
caaatgcgtg gttatcattt 20
<210> 48
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
tggcccctgc ccaccacg 18
<210> 49
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
agtgccgcag gctttcatcc ga 22
<210> 50
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tgcttgctag gccgcaagta cattc 25
<210> 51
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tttaggattt gtgttcacgc tca 23
<210> 52
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
ggaattttag gatttgtgtt cacg 24
<210> 53
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
aagaccaatc ttgtcacctc tgact 25
<210> 54
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
atttgtgttc acgctcaccg tg 22
<210> 55
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
ttaggatttg tgttcacgct ca 22
<210> 56
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
tcccatttag ggcattttgg 20
<210> 57
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tcccatttag ggcattttgg ac 22
<210> 58
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
tgagcgagga ctgcagcgta gacg 24
<210> 59
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
ccgtgcccag tgagcgagga ct 22
<210> 60
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
acgctcaccg tgcccagtga gc 22
<210> 61
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ggtttgctta agtagccatt gca 23
<210> 62
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
cagggccact tcgaagga 18
<210> 63
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
agcgaattga acaaccttcc a 21
<210> 64
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
tttggctggt gcttgtacca t 21
<210> 65
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
atccagggcc acttcgaagg 20
<210> 66
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
attttgtgtt tcaaccagcc tt 22
<210> 67
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
cggaggaagg ctgtttgtct a 21
<210> 68
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
ttcgcccttg ggaattctc 19
<210> 69
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
caccctcagt acgagtccta taacg 25
<210> 70
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
agggacgtaa acttaacacc actacc 26
<210> 71
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
cagtacgagt cctataacgg aggtc 25
<210> 72
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
cgtcaactta acaccactac cg 22
<210> 73
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
tgtcctctag ttcctggttg gcgttcc 27
<210> 74
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
cgtgacctca aagacatccc agagtgg 27
<210> 75
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
tttctactac ctcggaacag gacctcacgg 30
<210> 76
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
atctttttgg ttaccctgcg tt 22
<210> 77
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
gcaacatgcc cagcaagaag 20
<210> 78
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
cacctcagca gcaacatgc 19
<210> 79
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
ctgaactttt gaaagtgagc g 21
<210> 80
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
ccctgtaacg tttgtcagaa 20
<210> 81
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
gctgaacttt tgaaagtgag cg 22
<210> 82
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
cgtattttct tgcgctcgtc t 21
<210> 83
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
cagcgtgaac acccacctt 19
<210> 84
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
gcccatttgc ttttgcc 17
<210> 85
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
atgacgtgta cattcgtct 19
<210> 86
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
aagcggaccc caacc 15
<210> 87
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
aatgcagaag cgtgattgga agacgaatgt 30
<210> 88
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
ccgcgggctg gctgaacttt tg 22
<210> 89
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
acaccacacc aagatcggc 19
<210> 90
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
caaggcccac atctactaca agaa 24
<210> 91
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
gcacaagttc aaggcccaca tcta 24
<210> 92
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
gatccggtac atgtccatcg t 21
<210> 93
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
aggcccggga catgatccgg tacat 25
<210> 94
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
accaacgaca cctacaccaa g 21
<210> 95
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
cacaccaaga tcggcgccgc g 21
<210> 96
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
cgacgatgca gttgacgga 19
<210> 97
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
gcacgcggtc cgtgaa 16
<210> 98
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
tcctgcacgg ggacgggcac gc 22
<210> 99
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
ttcttgcgcg ccttgtg 17
<210> 100
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
cgggcccgct gttcttcttg c 21
<210> 101
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
acgatgcagt tgacggagg 19
<210> 102
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
cgacgatgca gttgacggag g 21
<210> 103
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
cgggcttcta ccacacgggc a 21
<210> 104
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
acgaccgtgt ggtccgggag c 21
<210> 105
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
tcggccctcg agcagcagga 20
<210> 106
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
ggcttctacc acacgggca 19
<210> 107
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
aaagaatttg gaagccatgc tc 22
<210> 108
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
tgccaacaag ggttctgtct t 21
<210> 109
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
tccctccgac ttcctctctt t 21
<210> 110
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
tgcacccctt cgctgact 18
<210> 111
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
aagccatgct ctcctacttc 20
<210> 112
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
tgagccaaca tagaaacaga ttgc 24
<210> 113
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
gctttagaaa aacagcatcg gaaa 24
<210> 114
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
cgttgttttg gacgaatttg c 21
<210> 115
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
ctccatcttg cctgcaggta ct 22
<210> 116
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
tttccagaat gttgagccaa 20
<210> 117
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
aaaccaggaa cactactc 18
<210> 118
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
cctccgactt cctct 15
<210> 119
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
cgatgctgtt tttctaaag 19
<210> 120
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
cggtgccgta ccgag 15
<210> 121
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
aaccaaattc cttcgctgtt ttt 23
<210> 122
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
ggtaaataca atggaaaatt caacga 26
<210> 123
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
atggaaaatt caacgaccgt ctta 24
<210> 124
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
tggaaaattc aacgaccgtc tta 23
<210> 125
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
cagaaccaaa ttccttcgct 20
<210> 126
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
gctgaacttc atctgggcta gct 23
<210> 127
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
acttgcgctt gctgcatcta g 21
<210> 128
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
acttgcgctt gctgcatct 19
<210> 129
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
ttttatcaag acttgcgctt gct 23
<210> 130
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
gctgaacttc atctgggc 18
<210> 131
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
aaagtgcccg tgaaat 16
<210> 132
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
cgtcttaact cgccaaat 18
<210> 133
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
ctcgccaaat ttagaatata 20
<210> 134
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
ccaaatttag aatatagcct agatg 25

Claims (22)

1. An oligonucleotide pair for detecting porcine pathogens comprising a first primer and a second primer, the pathogens selected from the group consisting of: classical swine fever virus, foot and mouth disease virus, encephalitis B virus, porcine reproductive and respiratory syndrome virus, swine influenza A virus, porcine epidemic diarrhea virus, porcine circovirus type 2, pseudorabies virus, porcine Selaginella virus, and mycoplasma hyopneumoniae, and is characterized in that:
when the pathogenic bacteria is classical swine fever virus, the sequence combination of the first primer and the second primer is selected from the following combinations: 1 and 7, 2 and 8, 5 and 8, 3 and 9, 4 and 10, 4 and 11, 6 and 10, and 6 and 11;
when the pathogenic bacteria are foot-and-mouth disease viruses, the sequence combination of the first primer and the second primer is selected from the following combinations: 16 and 22 SEQ ID NO, 17 and 23 SEQ ID NO, 20 and 23 SEQ ID NO, 18 and 24 SEQ ID NO, 20 and 24 SEQ ID NO, 19 and 25 SEQ ID NO, 19 and 26 SEQ ID NO, 21 and 25 SEQ ID NO, and 21 and 26 SEQ ID NO;
when the pathogenic bacteria is Japanese encephalitis virus, the sequence combination of the first primer and the second primer is selected from the following combinations: 31 and 35 of SEQ ID NO, 31 and 36 of SEQ ID NO, 32 and 35 of SEQ ID NO, 33 and 36 of SEQ ID NO, 34 and 35 of SEQ ID NO, and 34 and 36 of SEQ ID NO;
when the pathogenic bacteria are porcine reproductive and respiratory syndrome viruses, the sequence combination of the first primer and the second primer is selected from the following combinations: SEQ ID NO 40 and SEQ ID NO 44, SEQ ID NO 43 and SEQ ID NO 44, SEQ ID NO 40 and SEQ ID NO 45, SEQ ID NO 41 and SEQ ID NO 47, and SEQ ID NO 42 and SEQ ID NO 46;
when the pathogenic bacteria are swine influenza A viruses, the sequence combination of the first primer and the second primer is selected from the following combinations: SEQ ID NO 51 and SEQ ID NO 56, SEQ ID NO 54 and SEQ ID NO 56, SEQ ID NO 51 and SEQ ID NO 57, SEQ ID NO 52 and SEQ ID NO 56, SEQ ID NO 55 and SEQ ID NO 56, and SEQ ID NO 53 and SEQ ID NO 56;
when the pathogenic bacteria are porcine epidemic diarrhea viruses, the sequence combination of the first primer and the second primer is selected from the following combinations: SEQ ID NO 61 and 67, SEQ ID NO 62 and 68, SEQ ID NO 65 and 68, SEQ ID NO 63 and 69, SEQ ID NO 63 and 71, SEQ ID NO 64 and 70, SEQ ID NO 66 and 70, and SEQ ID NO 66 and 72;
when the pathogenic bacteria are porcine circovirus type 2, the sequence combination of the first primer and the second primer is selected from the following combinations: 77 and 82 SEQ ID NO, 78 and 83 SEQ ID NO, 79 and 84 SEQ ID NO, 81 and 84 SEQ ID NO, and 80 and 85 SEQ ID NO;
when the pathogenic bacteria is pseudorabies virus, the sequence combination of the first primer and the second primer is selected from the following combinations: SEQ ID NO. 89 and 96, SEQ ID NO. 90 and 97, SEQ ID NO. 91 and 98, SEQ ID NO. 92 and 99, SEQ ID NO. 93 and 100, SEQ ID NO. 94 and 101, and SEQ ID NO. 95 and 102;
when the pathogenic bacteria are porcine Saigra virus, the sequence combination of the first primer and the second primer is selected from the following combinations: 107 and 112 of SEQ ID NO, 107 and 116 of SEQ ID NO, 111 and 112 of SEQ ID NO, 111 and 116 of SEQ ID NO, 108 and 113 of SEQ ID NO, 109 and 114 of SEQ ID NO, and 110 and 115 of SEQ ID NO; and
when the pathogenic bacteria is mycoplasma hyopneumoniae, the sequence combination of the first primer and the second primer is selected from the following combinations: SEQ ID NO 121 and 126, SEQ ID NO 121 and 130, SEQ ID NO 125 and 126, SEQ ID NO 125 and 130, SEQ ID NO 122 and 127, SEQ ID NO 123 and 128, and SEQ ID NO 124 and 129.
2. The oligonucleotide pair of claim 1, further comprising an oligonucleotide probe having an oligonucleotide probe sequence, a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence-inhibiting molecule attached to a second position on the oligonucleotide probe.
3. The pair of oligonucleotides according to claim 2, wherein the pathogen is classical swine fever virus and the first and second primers are combined in SEQ ID No. 1 and SEQ ID No. 7, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between nucleotides 147 to 190 of the complete genomic sequence of the pathogenic classical swine fever virus;
the sequence combination of the first primer and the second primer is selected from the following combinations: 2 and 8, and 5 and 8, wherein the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between the 6482 th to 6515 th nucleotides of the complete genome sequence of the pathogenic classical swine fever virus;
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 3 and SEQ ID NO. 9, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 175 th to 219 th nucleotides of the complete genome sequence of the pathogenic classical swine fever virus; and
the sequence combination of the first primer and the second primer is selected from the following combinations: the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 139 th to 170 th nucleotides of the complete genome sequence of the pathogenic classical swine fever virus, and the oligonucleotide probe is composed of the oligonucleotide probes of SEQ ID NO. 4 and 10, SEQ ID NO. 4 and 11, SEQ ID NO. 6 and 10, and SEQ ID NO. 6 and 11.
4. The pair of oligonucleotides according to claim 2, wherein the pathogen is classical swine fever virus and the sequence of the first and second primers are set as SEQ ID No. 1 and SEQ ID No. 7, the sequence of the oligonucleotide probe is set as SEQ ID No. 12;
the sequence combination of the first primer and the second primer is selected from the following combinations: 2 and 8, 5 and 8, and the sequence of the oligonucleotide probe is shown as SEQ ID NO 13;
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 3 and SEQ ID NO. 9, the sequence of the oligonucleotide probe is shown as SEQ ID NO. 14; and
the sequence combination of the first primer and the second primer is selected from the following combinations: the sequence of the oligonucleotide probe is shown as SEQ ID NO. 15, and SEQ ID NO. 4 and 10, SEQ ID NO. 4 and 11, SEQ ID NO. 6 and 10, and SEQ ID NO. 6 and 11.
5. The pair of oligonucleotides of claim 2, wherein the pathogen is foot-and-mouth disease virus and the combination of sequences of the first and second primers is selected from the group consisting of: 16 and 22, 17 and 23, 20 and 23, 18 and 24, and 20 and 24, wherein the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between the 7907 th to 7949 th nucleotides of the complete genome sequence of pathogenic bacteria Foot and Mouth Disease Virus (FMDV); and
the sequence combination of the first primer and the second primer is selected from the following combinations: 19 and 25 of SEQ ID NO, 19 and 26 of SEQ ID NO, 21 and 25 of SEQ ID NO, and 21 and 26 of SEQ ID NO, wherein the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 8003 to 8031 nucleotides of the complete genome sequence of pathogenic Foot and Mouth Disease Virus (FMDV).
6. The pair of oligonucleotides of claim 2, wherein the pathogen is foot-and-mouth disease virus and the combination of sequences of the first and second primers is selected from the group consisting of: 16 and 22, 17 and 23, 20 and 23, 18 and 24, and 20 and 24, wherein the sequence of the oligonucleotide probe is selected from any one of SEQ ID NO 27, 28 and 29; and
the sequence combination of the first primer and the second primer is selected from the following combinations: the sequence of the oligonucleotide probe is shown as SEQ ID NO. 30, and the sequence of the oligonucleotide probe is shown as SEQ ID NO. 19 and SEQ ID NO. 25, SEQ ID NO. 19 and SEQ ID NO. 26, SEQ ID NO. 21 and SEQ ID NO. 25, and SEQ ID NO. 21 and SEQ ID NO. 26.
7. The pair of oligonucleotides of claim 2, wherein the pathogen is Japanese encephalitis virus and the sequence combination of the first and second primers is selected from the group consisting of: 31 and 35 of SEQ ID NO, 31 and 36 of SEQ ID NO, 32 and 35 of SEQ ID NO, 33 and 36 of SEQ ID NO, 34 and 35 of SEQ ID NO, and 34 and 36 of SEQ ID NO, wherein the oligonucleotide probe is an oligonucleotide of 13 to 25 base pairs between 3599 to 3626 nucleotides of the complete genome sequence of the pathogenic encephalitis B virus (JEV).
8. The pair of oligonucleotides of claim 2, wherein the pathogen is Japanese encephalitis virus and the sequence combination of the first and second primers is selected from the group consisting of: 31 and 35 of SEQ ID NO, 31 and 36 of SEQ ID NO, 32 and 35 of SEQ ID NO, 33 and 36 of SEQ ID NO, 34 and 35 of SEQ ID NO, and 34 and 36 of SEQ ID NO, wherein the sequence of the oligonucleotide probe is selected from any one of SEQ ID NO 37, 38 and 39.
9. The pair of oligonucleotides of claim 2, wherein the pathogen is porcine reproductive and respiratory syndrome virus and the combination of sequences of the first and second primers is selected from the group consisting of: 40 and 44, and 43 and 44, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 14532 th to 14554 th nucleotides of the complete genome sequence of the pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV);
the sequence combination of the first primer and the second primer is selected from the following combinations: 40 and 45, 41 and 45, and 41 and 47, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 14553 to 14584 nucleotides of the complete genome sequence of the pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV); and
when the sequences of the first primer and the second primer are combined into SEQ ID NO:42 and SEQ ID NO:46, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 14501 to 14537 nucleotides of the complete genome sequence of the pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
10. The pair of oligonucleotides of claim 2, wherein the pathogen is porcine reproductive and respiratory syndrome virus and the combination of sequences of the first and second primers is selected from the group consisting of: 40 and 44, 43 and 44, and the sequence of the oligonucleotide probe is shown as SEQ ID NO 48;
the sequence combination of the first primer and the second primer is selected from the following combinations: 40 and 45 of SEQ ID NO, 41 and 47 of SEQ ID NO, and the sequence of the oligonucleotide probe is shown as SEQ ID NO 49; and
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 42 and SEQ ID NO. 46, the sequence of the oligonucleotide probe is shown as SEQ ID NO. 50.
11. The pair of oligonucleotides of claim 2, wherein the pathogen is swine influenza a virus and the combination of sequences of the first and second primers is selected from the group consisting of: 51 and 56, 54 and 56, and 51 and 57, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between the 205 th to 237 th nucleotides of the Matrix Protein 1(Matrix Protein1) sequence of the pathogenic bacterium swine influenza a virus (SIV);
the sequence combination of the first primer and the second primer is selected from the following combinations: 52 and 56, and 55 and 56, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between the 200 th to the 239 th nucleotides of the matrix protein1 sequence of the pathogenic bacterium swine influenza a virus (SIV); and
when the sequences of the first primer and the second primer are combined into SEQ ID NO 53 and SEQ ID NO 56, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between the 169 th to the 239 th nucleotides of the matrix protein1 sequence of the pathogenic bacterium swine influenza A virus (SIV).
12. The pair of oligonucleotides of claim 2, wherein the pathogen is swine influenza a virus and the combination of sequences of the first and second primers is selected from the group consisting of: the sequence of the oligonucleotide probe is shown as SEQ ID NO. 58, and the sequence of the oligonucleotide probe is shown as SEQ ID NO. 51 and SEQ ID NO. 56, SEQ ID NO. 54 and SEQ ID NO. 56, and SEQ ID NO. 51 and SEQ ID NO. 57;
the sequence combination of the first primer and the second primer is selected from the following combinations: 52 and 56, 55 and 56, and the sequence of the oligonucleotide probe is shown as SEQ ID NO. 59; and
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 53 and SEQ ID NO. 56, the sequence of the oligonucleotide probe is shown as SEQ ID NO. 60.
13. The oligonucleotide pair of claim 2, wherein the pathogen is porcine epidemic diarrhea virus and the first and second primers are combined as SEQ ID NOs 61 and 67, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 156 th to 212 th nucleotides of the complete genome sequence of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV);
the sequence combination of the first primer and the second primer is selected from the following combinations: 62 and 68, and 65 and 68, the oligonucleotide probe being an oligonucleotide of 13 to 30 base pairs between the 27189 th to 27218 th nucleotides of the complete genomic sequence of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV);
the sequence combination of the first primer and the second primer is selected from the following combinations: 63 and 69, and 63 and 71, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between the 26588 th to 26625 th nucleotides of the complete genome sequence of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV); and
the sequence combination of the first primer and the second primer is selected from the following combinations: the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 22431 th to 22454 th nucleotides of the complete genome sequence of the pathogenic Porcine Epidemic Diarrhea Virus (PEDV).
14. The oligonucleotide pair of claim 2, wherein the pathogen is porcine epidemic diarrhea virus, and when the sequences of the first primer and the second primer are SEQ ID NO 61 and SEQ ID NO 67, the sequence of the oligonucleotide probe is shown as SEQ ID NO 73;
the sequence combination of the first primer and the second primer is selected from the following combinations: the sequence of the oligonucleotide probe is shown as SEQ ID NO 74;
the sequence combination of the first primer and the second primer is selected from the following combinations: the sequence of the oligonucleotide probe is shown as SEQ ID NO 75; and
the sequence combination of the first primer and the second primer is selected from the following combinations: the sequence of the oligonucleotide probe is shown as SEQ ID NO 76 in SEQ ID NO 64 and 70, SEQ ID NO 66 and 70, and SEQ ID NO 66 and 72.
15. The pair of oligonucleotides of claim 2, wherein the pathogen is porcine circovirus type 2 and the combination of sequences of the first and second primers is selected from the group consisting of: 77 and 82, 78 and 82, and 78 and 83, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between the 66 th to 94 th nucleotides of the complete genome sequence of the pathogenic porcine circovirus type 2 (PCV 2);
the sequence combination of the first primer and the second primer is selected from the following combinations: 79 and 84, and 81 and 84, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between the 523 th to 584 th nucleotides of the complete genome sequence of the pathogenic porcine circovirus type 2 (PCV 2); and
when the sequences of the first primer and the second primer are combined into SEQ ID NO:80 and SEQ ID NO:85, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between the 487 th nucleotide and the 546 th nucleotide of the complete genome sequence of the pathogenic porcine circovirus type 2 (PCV 2).
16. The pair of oligonucleotides of claim 2, wherein the pathogen is porcine circovirus type 2 and the combination of sequences of the first and second primers is selected from the group consisting of: 77 and 82 SEQ ID NO, 78 and 83 SEQ ID NO, wherein the sequence of the oligonucleotide probe is shown as SEQ ID NO 86;
the sequence combination of the first primer and the second primer is selected from the following combinations: 79 and 84 SEQ ID NO, 81 and 84 SEQ ID NO, and the sequence of the oligonucleotide probe is shown as 87 SEQ ID NO; and
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 80 and SEQ ID NO. 85, the sequence of the oligonucleotide probe is shown as SEQ ID NO. 88.
17. The pair of oligonucleotides of claim 2, wherein the pathogen is pseudorabies virus and the first and second primers are combined as SEQ ID NOs 89 and 96, the oligonucleotide probe is an oligonucleotide of 13 to 25 base pairs between 17998 to 18024 nucleotides of the complete genomic sequence of the pathogenic pseudorabies virus (PRV);
the sequence combination of the first primer and the second primer is selected from the following combinations: 90 and 97, and 91 and 98, said oligonucleotide probe being an oligonucleotide of 13 to 30 base pairs between nucleotides 18244 to 18298 of the complete genomic sequence of said pathogenic pseudorabies virus (PRV);
the sequence combination of the first primer and the second primer is selected from the following combinations: 92 and 99, and 93 and 100, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 16204 to 16225 nucleotides of the complete genome sequence of the pathogenic pseudorabies virus (PRV); and
the sequence combination of the first primer and the second primer is selected from the following combinations: 94 and 101, and 95 and 102, the oligonucleotide probe is an oligonucleotide of 13 to 25 base pairs between 18000 to 18030 nucleotides of the complete genome sequence of the pathogenic pseudorabies virus (PRV).
18. The oligonucleotide pair of claim 2, wherein the pathogen is pseudorabies virus and the sequences of the first and second primers are SEQ ID NO 89 and SEQ ID NO 96, the sequence of the oligonucleotide probe is shown in SEQ ID NO 103;
the sequence combination of the first primer and the second primer is selected from the following combinations: the sequence of the oligonucleotide probe is shown as SEQ ID NO. 104;
the sequence combination of the first primer and the second primer is selected from the following combinations: the sequence of the oligonucleotide probe is shown as SEQ ID NO. 105, and SEQ ID NO. 92 and 99, and SEQ ID NO. 93 and 100; and
the sequence combination of the first primer and the second primer is selected from the following combinations: the sequence of the oligonucleotide probe is shown as SEQ ID NO 106 in SEQ ID NO 94 and SEQ ID NO 101, and SEQ ID NO 95 and SEQ ID NO 102.
19. The pair of oligonucleotides of claim 2, wherein the pathogen is porcine Selaginella virus and the sequence combination of the first and second primers is selected from the group consisting of: 107 and 112, 107 and 116, 111 and 112, and 111 and 116, the oligonucleotide probe is an oligonucleotide of 13 to 25 base pairs between 7054 and 7079 nucleotides of the complete genome sequence of the pathogenic porcine Sambucus bulgaricus (SVVA);
when the sequences of the first primer and the second primer are combined into SEQ ID NO:108 and SEQ ID NO:113, the oligonucleotide probe is an oligonucleotide of 13 to 17 base pairs between 6933 and 6949 th nucleotides of the complete genome sequence of the pathogenic porcine Sakagavirus (SVVA);
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 109 and SEQ ID NO. 114, the oligonucleotide probe is an oligonucleotide of 13 to 21 base pairs between the 6953 th to 6973 th nucleotides of the complete genome sequence of the pathogenic porcine Sakay Valley Virus (SVVA); and
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 110 and SEQ ID NO. 115, the oligonucleotide probe is an oligonucleotide of 13 to 21 base pairs between 7154 to 7174 nucleotides of the complete genome sequence of the pathogenic porcine Sambucus Canadensis Valley virus (SVVA).
20. The pair of oligonucleotides of claim 2, wherein the pathogen is porcine Selaginella virus and the sequence combination of the first and second primers is selected from the group consisting of: 107 and 112 of SEQ ID NO, 107 and 116 of SEQ ID NO, 111 and 112 of SEQ ID NO, and 111 and 116 of SEQ ID NO, wherein the sequence of the oligonucleotide probe is shown as SEQ ID NO 117;
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 108 and SEQ ID NO. 113, the sequence of the oligonucleotide probe is shown as SEQ ID NO. 118;
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 109 and SEQ ID NO. 114, the sequence of the oligonucleotide probe is shown as SEQ ID NO. 119; and
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 110 and SEQ ID NO. 115, the sequence of the oligonucleotide probe is shown as SEQ ID NO. 120.
21. The pair of oligonucleotides of claim 2, wherein the pathogen is mycoplasma hyopneumoniae and the sequence combination of the first primer and the second primer is selected from the group consisting of: 121 and 126, 121 and 130, 125 and 126, and 125 and 130, wherein the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 215406 th to 215441 th nucleotides of the complete genome sequence of the pathogenic mycoplasma hyopneumoniae (Mhp);
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 122 and SEQ ID NO. 127, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 215742 th to 215772 th nucleotides of the complete genome sequence of the pathogenic mycoplasma hyopneumoniae (Mhp);
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 123 and SEQ ID NO. 128, the oligonucleotide probe is an oligonucleotide of 13 to 25 base pairs between 215750 th to 215774 th nucleotides of the complete genome sequence of the pathogenic mycoplasma hyopneumoniae (Mhp); and
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 124 and SEQ ID NO. 129, the oligonucleotide probe is an oligonucleotide of 13 to 30 base pairs between 215750 th to 215780 th nucleotides of the complete genome sequence of the pathogenic mycoplasma hyopneumoniae (Mhp).
22. The pair of oligonucleotides of claim 2, wherein the pathogen is mycoplasma hyopneumoniae and the sequence combination of the first primer and the second primer is selected from the group consisting of: 121 and 126 of SEQ ID NO, 121 and 130 of SEQ ID NO, 125 and 126 of SEQ ID NO, 125 and 130 of SEQ ID NO, and 125 and 130 of SEQ ID NO, wherein the sequence of the oligonucleotide probe is shown as SEQ ID NO 131;
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 122 and SEQ ID NO. 127, the sequence of the oligonucleotide probe is shown as SEQ ID NO. 132;
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 123 and SEQ ID NO. 128, the sequence of the oligonucleotide probe is shown as SEQ ID NO. 133; and
when the sequences of the first primer and the second primer are combined into SEQ ID NO. 124 and SEQ ID NO. 129, the sequence of the oligonucleotide probe is shown as SEQ ID NO. 134.
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CN101812539A (en) * 2009-12-03 2010-08-25 中国兽医药品监察所 Hog cholera virus TaqMan-MGB fluorescence quantitative RT-PCR differential detection kit and production method thereof
CN105886662A (en) * 2016-01-26 2016-08-24 金宇保灵生物药品有限公司 Method and special primer for rapidly breeding ST (swine testis) sensitive cell lines of hog cholera virus lapinized virus strain (C strain)
CN106282411A (en) * 2016-09-26 2017-01-04 湖南新南方养殖服务有限公司 A kind of Taqman real-time fluorescent PCR reagent case detecting pig Cord blood swine fever virus and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101812539A (en) * 2009-12-03 2010-08-25 中国兽医药品监察所 Hog cholera virus TaqMan-MGB fluorescence quantitative RT-PCR differential detection kit and production method thereof
CN105886662A (en) * 2016-01-26 2016-08-24 金宇保灵生物药品有限公司 Method and special primer for rapidly breeding ST (swine testis) sensitive cell lines of hog cholera virus lapinized virus strain (C strain)
CN106282411A (en) * 2016-09-26 2017-01-04 湖南新南方养殖服务有限公司 A kind of Taqman real-time fluorescent PCR reagent case detecting pig Cord blood swine fever virus and application thereof

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