CN102952850B - 用于检测结核分枝杆菌的实时荧光定量pcr方法、引物、探针及试剂盒 - Google Patents
用于检测结核分枝杆菌的实时荧光定量pcr方法、引物、探针及试剂盒 Download PDFInfo
- Publication number
- CN102952850B CN102952850B CN201110240765.9A CN201110240765A CN102952850B CN 102952850 B CN102952850 B CN 102952850B CN 201110240765 A CN201110240765 A CN 201110240765A CN 102952850 B CN102952850 B CN 102952850B
- Authority
- CN
- China
- Prior art keywords
- sample
- mycobacterium tuberculosis
- primer
- probe
- real
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000523 sample Substances 0.000 title claims abstract description 71
- 241000187479 Mycobacterium tuberculosis Species 0.000 title claims abstract description 44
- 229940010383 Mycobacterium tuberculosis Drugs 0.000 title claims abstract description 44
- 238000003753 real-time PCR Methods 0.000 title claims abstract description 19
- 238000011144 upstream manufacturing Methods 0.000 claims description 8
- 238000010791 quenching Methods 0.000 claims description 3
- 230000000171 quenching Effects 0.000 claims description 3
- 238000001514 detection method Methods 0.000 abstract description 27
- 229920003013 deoxyribonucleic acid Polymers 0.000 abstract description 23
- 238000006243 chemical reaction Methods 0.000 abstract description 20
- 206010036790 Productive cough Diseases 0.000 abstract description 10
- 210000003802 Sputum Anatomy 0.000 abstract description 10
- 201000010099 disease Diseases 0.000 abstract description 3
- 230000002265 prevention Effects 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances data:image/svg+xml;base64,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 data:image/svg+xml;base64,PD94bWwgdmVyc2lvbj0nMS4wJyBlbmNvZGluZz0naXNvLTg4NTktMSc/Pgo8c3ZnIHZlcnNpb249JzEuMScgYmFzZVByb2ZpbGU9J2Z1bGwnCiAgICAgICAgICAgICAgeG1sbnM9J2h0dHA6Ly93d3cudzMub3JnLzIwMDAvc3ZnJwogICAgICAgICAgICAgICAgICAgICAgeG1sbnM6cmRraXQ9J2h0dHA6Ly93d3cucmRraXQub3JnL3htbCcKICAgICAgICAgICAgICAgICAgICAgIHhtbG5zOnhsaW5rPSdodHRwOi8vd3d3LnczLm9yZy8xOTk5L3hsaW5rJwogICAgICAgICAgICAgICAgICB4bWw6c3BhY2U9J3ByZXNlcnZlJwp3aWR0aD0nODVweCcgaGVpZ2h0PSc4NXB4JyB2aWV3Qm94PScwIDAgODUgODUnPgo8IS0tIEVORCBPRiBIRUFERVIgLS0+CjxyZWN0IHN0eWxlPSdvcGFjaXR5OjEuMDtmaWxsOiNGRkZGRkY7c3Ryb2tlOm5vbmUnIHdpZHRoPSc4NS4wJyBoZWlnaHQ9Jzg1LjAnIHg9JzAuMCcgeT0nMC4wJz4gPC9yZWN0Pgo8dGV4dCB4PScxMy4zJyB5PSc1My42JyBjbGFzcz0nYXRvbS0wJyBzdHlsZT0nZm9udC1zaXplOjIzcHg7Zm9udC1zdHlsZTpub3JtYWw7Zm9udC13ZWlnaHQ6bm9ybWFsO2ZpbGwtb3BhY2l0eToxO3N0cm9rZTpub25lO2ZvbnQtZmFtaWx5OnNhbnMtc2VyaWY7dGV4dC1hbmNob3I6c3RhcnQ7ZmlsbDojRTg0MjM1JyA+SDwvdGV4dD4KPHRleHQgeD0nMjguMicgeT0nNjIuOScgY2xhc3M9J2F0b20tMCcgc3R5bGU9J2ZvbnQtc2l6ZToxNXB4O2ZvbnQtc3R5bGU6bm9ybWFsO2ZvbnQtd2VpZ2h0Om5vcm1hbDtmaWxsLW9wYWNpdHk6MTtzdHJva2U6bm9uZTtmb250LWZhbWlseTpzYW5zLXNlcmlmO3RleHQtYW5jaG9yOnN0YXJ0O2ZpbGw6I0U4NDIzNScgPjI8L3RleHQ+Cjx0ZXh0IHg9JzM1LjAnIHk9JzUzLjYnIGNsYXNzPSdhdG9tLTAnIHN0eWxlPSdmb250LXNpemU6MjNweDtmb250LXN0eWxlOm5vcm1hbDtmb250LXdlaWdodDpub3JtYWw7ZmlsbC1vcGFjaXR5OjE7c3Ryb2tlOm5vbmU7Zm9udC1mYW1pbHk6c2Fucy1zZXJpZjt0ZXh0LWFuY2hvcjpzdGFydDtmaWxsOiNFODQyMzUnID5PPC90ZXh0Pgo8L3N2Zz4K O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Chemical compound data:image/svg+xml;base64,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 data:image/svg+xml;base64,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 [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 8
- 238000007400 DNA extraction Methods 0.000 description 7
- 230000003321 amplification Effects 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 6
- 238000001556 precipitation Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 230000001580 bacterial Effects 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 230000000295 complement Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 108020004465 16S Ribosomal RNA Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000589248 Legionella Species 0.000 description 2
- 208000007764 Legionnaires' Disease Diseases 0.000 description 2
- 241000607768 Shigella Species 0.000 description 2
- 206010040550 Shigella infection Diseases 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 102000004965 antibodies Human genes 0.000 description 2
- 108090001123 antibodies Proteins 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000038129 antigens Human genes 0.000 description 2
- 108091007172 antigens Proteins 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003771 laboratory diagnosis Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000001717 pathogenic Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001954 sterilising Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 229940045505 Klebsiella pneumoniae Drugs 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 206010025482 Malaise Diseases 0.000 description 1
- 241001302239 Mycobacterium tuberculosis complex Species 0.000 description 1
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 1
- 229940076185 Staphylococcus aureus Drugs 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001186 cumulative Effects 0.000 description 1
- 201000009910 diseases by infectious agent Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drugs Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound data:image/svg+xml;base64,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 data:image/svg+xml;base64,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 [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound data:image/svg+xml;base64,<?xml version='1.0' encoding='iso-8859-1'?>
<svg version='1.1' baseProfile='full'
              xmlns='http://www.w3.org/2000/svg'
                      xmlns:rdkit='http://www.rdkit.org/xml'
                      xmlns:xlink='http://www.w3.org/1999/xlink'
                  xml:space='preserve'
width='300px' height='300px' viewBox='0 0 300 300'>
<!-- END OF HEADER -->
<rect style='opacity:1.0;fill:#FFFFFF;stroke:none' width='300.0' height='300.0' x='0.0' y='0.0'> </rect>
<path class='bond-0 atom-0 atom-1' d='M 172.9,145.6 L 198.0,165.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-0 atom-0 atom-1' d='M 180.6,143.5 L 198.1,157.2' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-28 atom-0 atom-19' d='M 172.9,145.6 L 143.5,157.6' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-30 atom-8 atom-0' d='M 177.3,114.1 L 172.9,145.6' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-1 atom-1 atom-2' d='M 198.0,165.1 L 227.5,153.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-2 atom-2 atom-3' d='M 227.5,153.1 L 231.8,121.7' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-2 atom-2 atom-3' d='M 221.8,147.6 L 224.9,125.5' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-3 atom-3 atom-4' d='M 231.8,121.7 L 241.3,117.8' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-3 atom-3 atom-4' d='M 241.3,117.8 L 250.8,114.0' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-6 atom-3 atom-7' d='M 231.8,121.7 L 206.7,102.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-4 atom-4 atom-5' d='M 270.9,117.2 L 278.6,123.2' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-4 atom-4 atom-5' d='M 278.6,123.2 L 286.4,129.2' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-5 atom-4 atom-6' d='M 262.8,98.8 L 264.2,88.5' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-5 atom-4 atom-6' d='M 264.2,88.5 L 265.6,78.2' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-7 atom-7 atom-8' d='M 206.7,102.1 L 177.3,114.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-7 atom-7 atom-8' d='M 204.7,109.8 L 184.1,118.2' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-8 atom-8 atom-9' d='M 177.3,114.1 L 169.5,108.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-8 atom-8 atom-9' d='M 169.5,108.1 L 161.8,102.1' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-9 atom-9 atom-10' d='M 141.7,98.8 L 132.2,102.7' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-9 atom-9 atom-10' d='M 132.2,102.7 L 122.7,106.5' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-9 atom-9 atom-10' d='M 141.2,105.9 L 134.6,108.6' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-9 atom-9 atom-10' d='M 134.6,108.6 L 128.0,111.3' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-10 atom-10 atom-11' d='M 122.7,106.5 L 97.6,87.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-31 atom-18 atom-10' d='M 118.4,138.0 L 122.7,106.5' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-11 atom-11 atom-12' d='M 97.6,87.0 L 68.2,99.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-11 atom-11 atom-12' d='M 95.6,94.7 L 75.0,103.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-12 atom-12 atom-13' d='M 68.2,99.0 L 60.5,93.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-12 atom-12 atom-13' d='M 60.5,93.0 L 52.7,87.0' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-15 atom-12 atom-16' d='M 68.2,99.0 L 63.8,130.5' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-13 atom-13 atom-14' d='M 44.6,68.6 L 46.0,58.3' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-13 atom-13 atom-14' d='M 46.0,58.3 L 47.5,48.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-14 atom-13 atom-15' d='M 32.6,83.7 L 23.1,87.6' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-14 atom-13 atom-15' d='M 23.1,87.6 L 13.6,91.4' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-16 atom-16 atom-17' d='M 63.8,130.5 L 88.9,150.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-16 atom-16 atom-17' d='M 71.5,128.4 L 89.1,142.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-17 atom-17 atom-18' d='M 88.9,150.0 L 118.4,138.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-18 atom-18 atom-19' d='M 118.4,138.0 L 143.5,157.6' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-18 atom-18 atom-19' d='M 126.0,135.9 L 143.6,149.6' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-19 atom-19 atom-20' d='M 143.5,157.6 L 139.1,189.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-20 atom-20 atom-21' d='M 139.1,189.0 L 164.2,208.6' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-20 atom-20 atom-21' d='M 139.0,197.0 L 156.5,210.7' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-29 atom-25 atom-20' d='M 109.6,201.0 L 139.1,189.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-21 atom-21 atom-22' d='M 164.2,208.6 L 159.8,240.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-22 atom-22 atom-23' d='M 159.8,240.1 L 130.4,252.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-22 atom-22 atom-23' d='M 153.0,236.0 L 132.4,244.3' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-23 atom-23 atom-24' d='M 130.4,252.0 L 105.3,232.5' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-24 atom-24 atom-25' d='M 105.3,232.5 L 109.6,201.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-24 atom-24 atom-25' d='M 112.2,228.7 L 115.3,206.6' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-25 atom-25 atom-26' d='M 109.6,201.0 L 84.5,181.5' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-26 atom-26 atom-27' d='M 84.5,181.5 L 76.3,184.8' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-26 atom-26 atom-27' d='M 76.3,184.8 L 68.1,188.2' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-27 atom-26 atom-28' d='M 86.4,181.3 L 86.2,179.4' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-27 atom-26 atom-28' d='M 86.2,179.4 L 86.0,177.6' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-27 atom-26 atom-28' d='M 82.6,181.7 L 82.5,179.8' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-27 atom-26 atom-28' d='M 82.5,179.8 L 82.3,178.0' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:2.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<text x='257.5' y='116.0' class='atom-4' style='font-size:12px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#4284F4' >N</text>
<text x='148.4' y='100.9' class='atom-9' style='font-size:12px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#E84235' >O</text>
<text x='157.1' y='95.8' class='atom-9' style='font-size:8px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#E84235' >+</text>
<text x='39.3' y='85.8' class='atom-13' style='font-size:12px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#4284F4' >N</text>
<text x='51.3' y='199.8' class='atom-27' style='font-size:12px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#E84235' >O</text>
<text x='60.1' y='194.7' class='atom-27' style='font-size:8px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#E84235' >-</text>
<text x='78.9' y='171.1' class='atom-28' style='font-size:12px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#E84235' >O</text>
</svg>
 data:image/svg+xml;base64,<?xml version='1.0' encoding='iso-8859-1'?>
<svg version='1.1' baseProfile='full'
              xmlns='http://www.w3.org/2000/svg'
                      xmlns:rdkit='http://www.rdkit.org/xml'
                      xmlns:xlink='http://www.w3.org/1999/xlink'
                  xml:space='preserve'
width='85px' height='85px' viewBox='0 0 85 85'>
<!-- END OF HEADER -->
<rect style='opacity:1.0;fill:#FFFFFF;stroke:none' width='85.0' height='85.0' x='0.0' y='0.0'> </rect>
<path class='bond-0 atom-0 atom-1' d='M 48.5,40.8 L 55.6,46.3' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-0 atom-0 atom-1' d='M 50.7,40.2 L 55.6,44.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-28 atom-0 atom-19' d='M 48.5,40.8 L 40.1,44.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-30 atom-8 atom-0' d='M 49.7,31.8 L 48.5,40.8' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-1 atom-1 atom-2' d='M 55.6,46.3 L 63.9,42.9' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-2 atom-2 atom-3' d='M 63.9,42.9 L 65.2,34.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-2 atom-2 atom-3' d='M 62.3,41.3 L 63.2,35.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-3 atom-3 atom-4' d='M 65.2,34.0 L 68.3,32.7' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-3 atom-3 atom-4' d='M 68.3,32.7 L 71.5,31.4' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-6 atom-3 atom-7' d='M 65.2,34.0 L 58.1,28.4' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-4 atom-4 atom-5' d='M 75.6,32.2 L 78.1,34.1' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-4 atom-4 atom-5' d='M 78.1,34.1 L 80.6,36.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-5 atom-4 atom-6' d='M 73.8,28.6 L 74.3,25.1' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-5 atom-4 atom-6' d='M 74.3,25.1 L 74.8,21.7' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-7 atom-7 atom-8' d='M 58.1,28.4 L 49.7,31.8' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-7 atom-7 atom-8' d='M 57.5,30.6 L 51.7,33.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-8 atom-8 atom-9' d='M 49.7,31.8 L 47.2,29.8' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-8 atom-8 atom-9' d='M 47.2,29.8 L 44.6,27.9' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-9 atom-9 atom-10' d='M 40.6,27.1 L 37.4,28.4' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-9 atom-9 atom-10' d='M 37.4,28.4 L 34.3,29.7' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-9 atom-9 atom-10' d='M 40.3,29.2 L 38.1,30.1' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-9 atom-9 atom-10' d='M 38.1,30.1 L 35.9,31.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-10 atom-10 atom-11' d='M 34.3,29.7 L 27.2,24.2' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-31 atom-18 atom-10' d='M 33.0,38.6 L 34.3,29.7' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-11 atom-11 atom-12' d='M 27.2,24.2 L 18.8,27.5' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-11 atom-11 atom-12' d='M 26.6,26.3 L 20.7,28.7' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-12 atom-12 atom-13' d='M 18.8,27.5 L 16.3,25.6' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-12 atom-12 atom-13' d='M 16.3,25.6 L 13.7,23.6' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-15 atom-12 atom-16' d='M 18.8,27.5 L 17.6,36.5' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-13 atom-13 atom-14' d='M 12.0,20.0 L 12.5,16.5' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-13 atom-13 atom-14' d='M 12.5,16.5 L 12.9,13.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-14 atom-13 atom-15' d='M 9.7,22.8 L 6.5,24.1' style='fill:none;fill-rule:evenodd;stroke:#4284F4;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-14 atom-13 atom-15' d='M 6.5,24.1 L 3.4,25.4' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-16 atom-16 atom-17' d='M 17.6,36.5 L 24.7,42.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-16 atom-16 atom-17' d='M 19.8,35.9 L 24.7,39.7' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-17 atom-17 atom-18' d='M 24.7,42.0 L 33.0,38.6' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-18 atom-18 atom-19' d='M 33.0,38.6 L 40.1,44.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-18 atom-18 atom-19' d='M 35.2,38.0 L 40.2,41.9' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-19 atom-19 atom-20' d='M 40.1,44.1 L 38.9,53.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-20 atom-20 atom-21' d='M 38.9,53.1 L 46.0,58.6' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-20 atom-20 atom-21' d='M 38.9,55.3 L 43.8,59.2' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-29 atom-25 atom-20' d='M 30.6,56.5 L 38.9,53.1' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-21 atom-21 atom-22' d='M 46.0,58.6 L 44.8,67.5' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-22 atom-22 atom-23' d='M 44.8,67.5 L 36.4,70.9' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-22 atom-22 atom-23' d='M 42.9,66.4 L 37.0,68.7' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-23 atom-23 atom-24' d='M 36.4,70.9 L 29.3,65.4' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-24 atom-24 atom-25' d='M 29.3,65.4 L 30.6,56.5' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-24 atom-24 atom-25' d='M 31.3,64.3 L 32.2,58.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-25 atom-25 atom-26' d='M 30.6,56.5 L 23.5,50.9' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-26 atom-26 atom-27' d='M 23.5,50.9 L 20.8,52.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-26 atom-26 atom-27' d='M 20.8,52.0 L 18.1,53.1' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-27 atom-26 atom-28' d='M 24.0,50.9 L 23.9,50.0' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-27 atom-26 atom-28' d='M 23.9,50.0 L 23.8,49.2' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-27 atom-26 atom-28' d='M 22.9,51.0 L 22.8,50.2' style='fill:none;fill-rule:evenodd;stroke:#3B4143;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<path class='bond-27 atom-26 atom-28' d='M 22.8,50.2 L 22.7,49.3' style='fill:none;fill-rule:evenodd;stroke:#E84235;stroke-width:1.0px;stroke-linecap:butt;stroke-linejoin:miter;stroke-opacity:1' />
<text x='71.7' y='33.6' class='atom-4' style='font-size:6px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#4284F4' >N</text>
<text x='40.8' y='29.3' class='atom-9' style='font-size:6px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#E84235' >O</text>
<text x='45.0' y='26.9' class='atom-9' style='font-size:3px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#E84235' >+</text>
<text x='9.9' y='25.0' class='atom-13' style='font-size:6px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#4284F4' >N</text>
<text x='13.3' y='57.3' class='atom-27' style='font-size:6px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#E84235' >O</text>
<text x='17.4' y='54.9' class='atom-27' style='font-size:3px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#E84235' >-</text>
<text x='21.1' y='49.2' class='atom-28' style='font-size:6px;font-style:normal;font-weight:normal;fill-opacity:1;stroke:none;font-family:sans-serif;text-anchor:start;fill:#E84235' >O</text>
</svg>
 C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissues Anatomy 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
Abstract
本发明公开了一种用于检测结核分枝杆菌的实时荧光定量PCR方法，包括步骤：1)采集患者痰液样本并进行处理；2)提取样本的DNA；3)以标准菌株的DNA为阳性对照，灭菌水为阴性对照，进行样本DNA的实时荧光定量PCR反应；4)根据反应得到的Ct值判断样本的检测结果，并计算样本中结核分枝杆菌的拷贝数。本发明还公开了上述检测方法所用的引物和探针以及包含该引物和探针的试剂盒。该检测方法简便、快速且特异性高，可以实现临床痰液样本中结核分枝杆菌的快速定量检测，从而有助于疾病的预防和及时治疗。
Description
技术领域
[0001] 本发明涉及生物医学领域，特别是涉及一种用于检测结核分枝杆菌的荧光定量 PCR方法。本发明还涉及用于该方法的引物和探针，以及包含该引物和探针的试剂盒。
背景技术
[0002] 结核病是全球面临的主要公共健康问题之一，是引起成年人死亡的主要传染源， 尤其是近年来，由于大规模人口流动及细菌本身耐药性的产生，结核病的发病率有回升的 趋势。
[0003] 结核分枝杆菌（MTB)是结核病的病原体，目前对MTB的诊断主要采用实验室诊断 与临床体征相结合的方法。实验室诊断主要包括涂片染色法、细菌培养法、抗原抗体检测、 PCR等方法。其中，病人组织或体液标本的细菌培养是目前检测结核分枝杆菌的金标准，但 其缺点是检测周期较长（需要8周），难以实现致病菌的快速检测，因此在临床上很容易贻 误病情，不利于早期治疗。抗原抗体检测通常采用ELISA(酶联免疫吸附试验）法，该方法 具有操作简单、价格低廉的优点，但在检测特异性和灵敏度方面还有待提高。
[0004] 随着分子生物学的发展，PCR技术已成为目前常见的实验手段，特别是实时荧光定 量PCR，因具有技术成熟、灵敏度高、操作简单和特异性好的优点，已被逐步应用到临床上。 同时，利用基因芯片在细菌耐药性方面的应用，近几年出现了不少将荧光定量PCR技术和 基因芯片相结合的检测MTB及其耐药基因的方法，例如，名称为"一种从痰中提取细菌核酸 的方法、试剂盒及其应用"的中国发明专利（申请号200810105172. X)，利用了基因芯片技 术，通过比较不同菌株中16S rRNA基因的差异，设计引物和探针对MTB进行检测，但由于 16S基因较短（1500bp)，因此该方法的检测特异性较低；另一名称为"一种结核分枝杆菌复 合群的荧光定量RT-PCR检测方法"的中国发明专利申请（申请号201010522800. 1)，在引物 和探针设计时，也采取了比较不同菌株中16S rRNA基因的方法，因此，也存在上述的问题， 此外，该方法采用人的基因组DNA最为阴性对照进行检测，其检测结果的特异性有待改进。
发明内容
[0005] 本发明要解决的技术问题之一是提供一种能够特异性检测结核分枝杆菌的引物 对。
[0006] 为解决上述技术问题，本发明的用于实时荧光定量PCR检测结核分枝杆菌的引物 对，包括上游引物和下游引物，其中，上游引物具有如SEQ ID No :1所示的序列或其互补链， 下游引物具有如SEQ ID No :2所示的序列或其互补链；所述上游引物还可以向5'端方向延 伸10个碱基，所述下游引物还可以向3'端方向延伸10个碱基。
[0007] 本发明要解决的技术问题之二是提供一种能够特异性检测结核分枝杆菌的探针。
[0008] 为解决上述技术问题，本发明的用于实时荧光定量PCR检测结核分枝杆菌的探 针，具有如SEQ ID No :3所示的序列或其互补链，且所述探针的5'端连接有一个荧光报告 基团，3'端连接有一个荧光淬灭基团。所述探针还可以向3'端方向延伸10个碱基。
[0009] 本发明要解决的技术问题之三是提供一种利用上述引物对和探针进行实时荧光 定量PCR检测结核分枝杆菌的方法，它不仅简便、快速，而且特异性高。
[0010] 为解决上述技术问题，本发明的用于检测结核分枝杆菌的实时荧光定量PCR方 法，包括以下步骤：
[0011] 1)采集患者痰液样本，并进行处理；
[0012] 2)提取样本中的DNA ;
[0013] 3)以结核分枝杆菌标准菌株中提取的DNA为阳性对照，灭菌水为阴性对照，利用 前述引物对和探针，对样本DNA进行实时荧光定量PCR反应，测定样本和标准品的Ct值；
[0014] 4)利用步骤3)的测定结果，判断样本中是否含有结核分枝杆菌，并计算样本中结 核分枝杆菌的拷贝数。
[0015] 步骤1)采集的样本，在2~8°C下保存不应超过24小时，在-20°C下保存不超过 3个月，在_80°C下可以长期保存，样本应低温运输。
[0016] 步骤1)中，可以按照如下步骤进行样本的处理：取2~3mL样本，加入1~4倍体 积的4%氢氧化钠，混匀，室温液化0. 5~3小时，至样本变为透明溶液；取已液化的痰液样 本1~I. 5mL，放入灭菌的I. 5mL灭菌PE管中，10, 000~15, OOOrpm离心5~10分钟，弃 上清；沉淀加入ImL生理盐水重悬，10, 000~15, OOOrpm离心5~10分钟，弃上清；沉淀用 于进行DNA的提取。
[0017] 步骤2)中，样本DNA的提取，可以利用商业化的DNA提取试剂盒，例如Qiagen公司 或TaKaRa公司的DNA提取试剂盒，按照试剂盒的说明书进行操作；也可以按照以下方法进 行操作：向经步骤1)处理后得到的沉淀中，加入50~100 μ L灭菌水，放在KKTC水浴中煮 沸10~15min，所得溶液即为样本DNA的溶液，取上清2~4 μ L，可直接用于荧光定量PCR 反应。
[0018] 步骤3)中，PCR反应采用20 μ L的反应体系，每份反应液中含2 XPCR Master Mix 10 yL，10 ymol/L 的上、下游引 物各 0· 2 ~0· 6 yL，10 ymol/L 探针 0· 6 ~I. 0 yL，ROX ReferenceDye (50 X) 0. 4 μ L，结核分枝杆菌DNA模板1~3 μ L，用灭菌水补充至总体积为 20. 0 μ L，反应溶液在冰上配制。PCR反应条件为：95°C，30秒；然后进行40个循环：95°C，5 秒，60°C，31 秒。
[0019] 本发明要解决的技术问题之四是提供一种用于实时荧光定量PCR检测结核分枝 杆菌的试剂盒。
[0020] 为解决上述技术问题，本发明的用于实时荧光定量PCR检测结核分枝杆菌的试剂 盒，除包含有常规实时荧光定量PCR试剂（包括：Taq酶、dNTP试剂、PCR缓冲液、灭菌水 等）、阳性对照（结核分枝杆菌标准菌株的DNA，浓度分别为10 6、105、104C〇pieS/mL)、阴性对 照外，还包含有上述引物对和探针。
[0021] 本发明的用于检测结核分枝杆菌的实时荧光定量PCR方法，以及用于该方法的引 物和探针或试剂盒，不仅对结核分枝杆菌具有较好的检测特异性，而且简便、快速，可以在 2~3小时内完成临床痰液样本的检测诊断，从而有助于疾病的预防和及时诊断，在临床上 具有良好的应用前景。
附图说明
[0022]图1是结核分枝杆菌检测灵敏度测试图，图中横坐标是PCR反应的循环数，纵坐标 是采集的荧光信号值；
[0023] 图2是采用琼脂糖凝胶电泳验证本发明试剂盒的可靠性；
[0024] 图3A是使用本发明的试剂盒进行志贺菌检测的结果；
[0025] 图3B是使用本发明的试剂盒进行军团菌检测的结果；
[0026] 图3C是使用本发明的试剂盒进行其他菌检测的结果；
[0027] 图4是利用本发明的试剂盒进行临床痰液样本检测的结果。
具体实施方式
[0028] 下面结合附图和实施例，对本发明作进一步详细的说明。
[0029] 以下实施例仅用于说明本发明，而不用于限制本发明的范围。实施例中未注明具 体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册（New York :ColdSpring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建 议的条件。
[0030] 1.样本的采集和处理，及样本DNA的提取
[0031] 按照临床样本采集要求，采集患者的痰液样本，采集的样本保存在_20°C。
[0032] 将样本在冰上融化，取样本2mL，向样本中加入4mL的4%的NaOH溶液，混匀，室温 处理0. 5小时。然后，取样本I. 5mL，在13, OOOrpm离心10分钟，弃上清，加入ImL的IXPBS 重悬，在13, OOOrpm离心10分钟，弃上清，保留沉淀。加入50 μ L灭菌水，在100°C水浴中煮 沸10分钟，所得溶液即为含样本DNA的溶液，取1 μ L该溶液用于实时荧光定量PCR反应。
[0033] 2.检测灵敏度测试
[0034] 通过对GenBank上结核分枝杆菌和其他物种序列的比对，选取结核分枝杆菌特异 序列，设计出一组用于PCR检测结核分枝杆菌核苷酸片段的引物和探针，其序列如下：
[0035] 上游引物：5 ' -GGCTGTCGTTTTGCTCTG-3 '
[0036] 下游引物：5 ' -CTGATGTTGGTGTTGTAGGC-3 '
[0037] 检测探针：5' -CGACACCCGAACAACAGAGC-3'
[0038] 上述检测探针的5'端连接有一个荧光报告基团FAM(6-羧基荧光素），3'端连接 有一个荧光淬灭基团TAMRA (6-羧基四甲基丹诺明）。
[0039] PCR反应采用20 μ L的反应体系，每份反应液中含有2XPCR Master Mix 10 μ L，上、下游引物（10 μ mol/L)各 0· 4 μ L，探针（10 μ mol/L) 0· 8 μ L，ROX Reference Dye (50X) 0· 4 μ L，灭菌水6· 0 μ L，结核分枝杆菌DNA样品（浓度分别为：（a) 2X 10°ng/ μ L， (b) 2 X 10 1Iig/ μ L，（c) 2 X 10 2ng/ μ L，（d) 2 X 10 3ng/ μ L，（e) 2 X 10 4ng/ μ L，（f) 2 X 10 5ng/ μ L，（g) 2X l(T6ng/ μ L，（h) 2X l(T7ng/ μ L，（i) 2X l(T8ng/ μ L) 2 μ L。反应溶液在冰上配制， 配制完成后使用ABI 7300real-Time PCR System进行PCR反应。
[0040] 反应条件：95°C，30秒；然后进行40个循环，每个循环为：95°C，5秒，60°C，31秒。
[0041] 反应结束后，对扩增曲线进行分析，得到以循环数为横坐标，吸光值为纵坐标的定 量曲线，如图1所示。从图中可以看出，当DNA模板浓度分别为2X10°ng/μ LdXKT1Iig/ μ L，2 X 10_2ng/ μ L，2 X 10_3ng/ μ L，2 X 10_4ng/ μ L时，均出现明显的扩增曲线，但当模板浓 度低于2X l(T4ng/ μ L时，未出现明显的扩增曲线。即使用本发明的荧光定量PCR方法及试 剂盒对结核分枝杆菌的检测可以达到2XKT4ng/yL(基因组DNA)的检测灵敏度，相当于40 拷贝数（copies)的结核分枝杆菌。
[0042] 为了验证本发明试剂盒检测结果的可靠性，对实时荧光定量PCR反应产物进行琼 脂糖凝胶电泳检测，结果如图2所示，其中的泳道1~9为上述不同拷贝数的结核分枝杆菌 DNA模板，泳道10为阴性对照（灭菌水），M为IOObp标记物（Marker)。检测结果与目的片 段大小一致。
[0043] 3.本发明试剂盒的特异性检验
[0044] 分别用结核分枝杆菌DNA提取物、志贺菌DNA提取物、大肠杆菌DNA提取物、肺炎 克雷伯菌DNA提取物、李斯特菌DNA提取物、肠炎沙门菌DNA提取物、金黄色葡萄球菌DNA 提取物、军团菌DNA提取物为模板，采用本发明的试剂盒，按照上述检测灵敏度测试中所述 的反应体系和条件，进行实时荧光定量PCR检测。检测结果如图3A~3C所示，从图中可以 看出，只有以结核分枝杆菌DNA为模板的样品有明显的扩增曲线，其他的样本均无明显的 扩增曲线。
[0045] 4.临床痰液样本的检测
[0046] 按照临床样本采集要求，采集患者痰液样本，采集的样本保存在2~8°C (不超过 4小时）。
[0047] 取样本2mL，向样本中加入4mL的4% NaOH溶液，混勾，室温处理I. 0小时。然后取 样本I. 5mL，在13, OOOrpm离心10分钟，弃上清，加入I. 5mL的IXPBS重悬，在13, OOOrpm 离心10分钟，弃上清，保留沉淀。加入50 μ L灭菌水，放在100°C水浴中煮沸10分钟，所得 溶液即为样本DNA的溶液，可直接用于进行荧光定量PCR反应。
[0048] 取上清2 μ L，以结核分枝杆菌标准菌株（源自ATCC27294,美国标准菌种保藏中 心）提取的DNA为阳性对照，无菌蒸馏水为阴性对照，利用本发明的试剂盒，并按照上述检 测灵敏度测试中所述的反应体系和条件进行实时荧光定量PCR反应。
[0049] 反应结束后，分析PCR检测结果，得到每个样本的Cti^值、阳性标准品的值 以及阴性对照的Ct水值（Ct为域值循环数，且Ct标准彡35, Ct水> 35)，若Ct样本值彡35,且 呈现典型的扩增曲线，则为阳性结果，表明样本中含有结核分枝杆菌；若Ctiws值> 35,或者 无扩增信号，则为阴性结果，表明样本中无结核分枝杆菌。根据图4所示的反应曲线可以看 出，阳性标准品和临床采集的样本均呈现出明显的扩增曲线，表明该临床痰液样本中含有 结核分枝杆菌。进一步根据公式copies =标准品拷贝数*2，式中ACt = Ct样本-Ct标准， 可以计算得到样本中所含结核分枝杆菌的拷贝数。
Claims (3)
1. 一种用于实时荧光定量PCR检测结核分枝杆菌的引物对，其特征在于，该引物对包 括上游引物和下游引物，所述上游引物的序列如SEQ ID No: 1所示；所述下游引物的序列如 SEQ ID No:2 所示。
2. -种用于实时荧光定量PCR检测结核分枝杆菌的探针，其特征在于，该探针的序列 如SEQ ID No: 3所示；且该探针的5'端连接有一个荧光报告基团，3'端连接有一个荧光淬 灭基团。
3. -种用于实时荧光定量PCR检测结核分枝杆菌的试剂盒，其特征在于，包含有权利 要求1所述的引物对和权利要求2所述的探针。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110240765.9A CN102952850B (zh) | 2011-08-22 | 2011-08-22 | 用于检测结核分枝杆菌的实时荧光定量pcr方法、引物、探针及试剂盒 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110240765.9A CN102952850B (zh) | 2011-08-22 | 2011-08-22 | 用于检测结核分枝杆菌的实时荧光定量pcr方法、引物、探针及试剂盒 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102952850A CN102952850A (zh) | 2013-03-06 |
| CN102952850B true CN102952850B (zh) | 2015-05-20 |
Family
ID=47762233
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110240765.9A Active CN102952850B (zh) | 2011-08-22 | 2011-08-22 | 用于检测结核分枝杆菌的实时荧光定量pcr方法、引物、探针及试剂盒 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102952850B (zh) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004837B (zh) * | 2014-05-20 | 2015-07-15 | 广州海力特生物科技有限公司 | 检测结核分枝杆菌的pcr引物、引物组、探针及其试剂盒和检测方法 |
| CN106811529A (zh) * | 2017-02-27 | 2017-06-09 | 苏州百源基因技术有限公司 | 结核分枝杆菌的荧光定量pcr检测试剂盒及引物、探针 |
| CN106755533A (zh) * | 2017-02-28 | 2017-05-31 | 新乡医学院第附属医院 | 一种结核杆菌的检测方法 |
| CN108384867A (zh) * | 2018-04-28 | 2018-08-10 | 宁波市鄞州人民医院 | 一种实时荧光pcr检测下呼吸道细菌特异性基因的引物、探针、方法及试剂盒 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108398A (zh) * | 2010-12-01 | 2011-06-29 | 广东省中医院 | 结核分枝杆菌荧光定量pcr检测方法 |
-
2011
- 2011-08-22 CN CN201110240765.9A patent/CN102952850B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108398A (zh) * | 2010-12-01 | 2011-06-29 | 广东省中医院 | 结核分枝杆菌荧光定量pcr检测方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102952850A (zh) | 2013-03-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Timms et al. | How accurately can we detect Mycobacterium avium subsp. paratuberculosis infection? | |
| Suchodolski et al. | Application of molecular fingerprinting for qualitative assessment of small-intestinal bacterial diversity in dogs | |
| CN103866034A (zh) | 一种胃液中幽门螺杆菌多重实时荧光定量pcr检测试剂盒及检测方法 | |
| CN102363815B (zh) | 运用交叉引物核酸恒温扩增技术检测沙门菌的试剂及其扩增方法和检测方法 | |
| CN102952850B (zh) | 用于检测结核分枝杆菌的实时荧光定量pcr方法、引物、探针及试剂盒 | |
| CN102108398B (zh) | 结核分枝杆菌荧光定量pcr检测方法 | |
| CN102409102B (zh) | 一种鉴别牛分枝杆菌的pcr引物及方法 | |
| CN108866164A (zh) | B族链球菌的检测方法及试剂盒 | |
| Wainø et al. | Identification of campylobacteria isolated from Danish broilers by phenotypic tests and species‐specific PCR assays | |
| CN103014135B (zh) | 一种鉴定结核分枝杆菌的方法 | |
| CN102533959A (zh) | 一种鉴别结核分枝杆菌的多重pcr试剂盒 | |
| CN104263842A (zh) | 一种鱼源无乳链球菌的荧光定量pcr检测方法 | |
| CN103333903A (zh) | 检测幽门螺杆菌的靶序列、引物和探针及其试剂盒 | |
| Shaheen et al. | Detection of fluoroquinolone resistance level in clinical canine and feline Escherichia coli pathogens using rapid real-time PCR assay | |
| CN102146478B (zh) | 一种用于鉴定类鼻疽伯克霍尔德菌的pcr试剂盒 | |
| CN101586157A (zh) | 一种鱼类病原菌海豚链球菌分子诊断试剂盒及检测方法 | |
| CN102080125B (zh) | 用于检测鉴定伤寒沙门氏菌核酸片段的引物、探针及芯片 | |
| CN108411014A (zh) | 鉴别a型和b型牛多杀性巴氏杆菌的双重实时荧光定量pcr的引物和探针以及检测方法 | |
| CN105506072B (zh) | 检测桑丁香假单胞菌的引物及其应用 | |
| CN102656277A (zh) | 用于检测结核分枝杆菌的方法和/或引物 | |
| CN102057055A (zh) | 分支杆菌的快速检测 | |
| CN103509859B (zh) | 山羊结核病pcr检测试剂盒 | |
| CN100415900C (zh) | 淋病奈瑟菌荧光定量pcr检测试剂盒 | |
| CN103509790B (zh) | 利用单管巢式实时聚合酶链反应(pcr)的改良结核菌诊断方法 | |
| CN104611420A (zh) | 一种结核菌检测试剂盒 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| C06 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| C10 | Entry into substantive examination | ||
| GR01 | Patent grant | ||
| C14 | Grant of patent or utility model |