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GB∕T 38164-2019 常见畜禽动物源性成分检测方法 实时荧光PCR法_第1页
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犐犆犛07.080

犃40

???????????

犌犅/犜38164—2019

??????????????

????犘犆犚?

犐犱犲狀狋犻犳犻犮犪狋犻狅狀狅犳犪狀犻犿犪犾犻狀犵狉犲犱犻犲狀狋犳狉狅犿犮狅犿犿狅狀犾犻狏犲狊狋狅犮犽犪狀犱狆狅狌犾狋狉狔—

犚犲犪犾狋犻犿犲犘犆犚

20191018??20191018??

??????????

????????????

??

??

?????GB/T1.1—2009???????。

??????????????????(SAC/TC387)?????。

???????:???????????、????、????、????、????。

????????:??、???、???、???、???、??、??、???、???、???、

???、???。

犌犅/犜38164—2019

??????????????

????犘犆犚?

1??

?????????????????????PCR????。

???????????、??、?、???????、??、??、??、??、?、??、??、??

?、??、?、?、?、?、?、??、??、??、?、??、??、?、??????Taqman??????

PCR????。

???(LOD)1%(????)。

2???????

??????????????????。??????????,????????????

?。???????????,?????(????????)??????。

GB/T6682??????????????

GB/T27403—2008??????????????????

GB/T34796?????????????????????

GB/T35918?????????????????Sanger???

3?????、

???

3.1?????

?????????????。

3.1.1

????犘犆犚狉犲犪犾狋犻犿犲犘犆犚

?PCR???????????,???????????????PCR????。

3.1.2

犆狋?犮狔犮犾犲狋犺狉犲狊犺狅犾犱

??????????????????????????。

3.2???

???????????。

ATP8:ATP?????8(ATPsynthasesubunit8)

COⅠ:????C?????Ⅰ(CytochromeCoxidaseⅠ)

CTAB:??????????(Cetyltrithylammoniumbromide)

犮狔狋犫:????b(Cytochromeb)

DNA:??????(Deoxyribonucleicacid)

Dloop:D??(Displacementloopregion)

EDTA:??????(Ethylenediaminetetraaceticacid)

犌犅/犜38164—2019

Na2EDTA:????????(Ethylenediaminetetraaceticaciddisodiumsalt)

ND1:?????????????????1(Nicotinamideadeninedinucleotideoxidoreductase

subunit1)

犜犪狇

DNA???:??DNA???(

犜犪狇

DNApolymerase)

Tris:????????(Trishydroxymethylaminomethane)

4??

???????????????????????????PCR??,??PCR?????

???????????????,??????????????????。

5????????

???????????????????????????1。

?1??????

??????????

????

F:TCTGCCCTATCAACTTTCGATGGTA

R:AATTTGCGCGCCTGCTGCCTTCCTT

P:5′FAMCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACC3′TAMRA

18SrRNA??

??

F:TGGAGTAATCCTTCTGCTCACAGT

R:TGACTGTTGCTCCTCAGAATGATA

P:5′FAMAGCATTTATAGGATACGTCC3′MGBNFG

犮狔狋犫??

??

F:GACTAATATTCGAAAATCC

R:CTCCTAGGAGGGAGCCGAAGTTTCAC

P:5′FAMCAACGCATTCATTGACCTTCCAGCTCCAT3′TAMRA

犮狔狋犫??

??

F:TTCATTGAYCTCCCTGCTCC

R:GGAATAGGCCGGTGAGGATT

P:5′FAMACTTTGGCTCTCTCC3′MGBNFG

ATP8??

??

F:ATTCTTTGCCTTCCACTTCA

R:AGCGTATGCGAATAGGAAAT

P:5′FAMCCTACACGAAACAGGCTCTAATAACCCGAC3′TAMRA

犮狔狋犫??

???

F:CCTACCACCGAAGCAATAGTGG

R:GGTGCACTCTCAATAACTTCTGAGA

P:5′FAMCACTGTAGCTACTCAAGAAGC3′TAMRA

COⅠ??

??

F:TAGTTATGTAAACAAGACTG

R:ATAGACTGTAGTAGCAAGGAA

P:5′FAMAGGACTTGGCGGTGCTTTAT3′TAMRA

12SrRNA??

??

F:CTCCCAGTACGAAAGGACCA

R:GGGCGGGATATGTTTGTGTA

P:5′FAMCCAACTTTAATCAAGCGCCT3′TAMRA

16SrRNA??

犌犅/犜38164—2019

?1(?)

??????????

??

F:TGGAGTAATCCTCCTGCTC

R:GGATTGCTGAAAGAAGATTA

P:5′FAMCATAGGCTATGTTTTACCATGAG3′TAMRA

犮狔狋犫??

??

F:CGGAGTAATCCTCCTATTTGC

R:CTAGGCTTGTGCCAATATATGGA

P:5′FAMTATTACCAACCTCCTTT3′MGBNFG

犮狔狋犫??

??

F:GAGCAACCCCTCAACGTACT

R:ACCTCCTCGTCGATATGGACTC

P:5′FAMCCCCTCAACGTACTAACAGTAACCAGAC3′TAMRA

16SrRNA??

?

F:GGCCACACAAATCCTCACAG

R:TGTGTTGGCTACTGAGGAGAAA

P:5′FAMCCTACTGGCTATGCACTACACCGCAGAC3′Eclipse

犮狔狋犫??

?

F:CCACCGCGAATCCTCAAT

R:TCTTGTTTTTGGAGGATTTGTTCTC

P:5′FAMCCATCGCAGAGCCTGGTCGATATTTTT3′Eclipse

16SrRNA??

??

F:GGATCACTTTGCCCACTTTAA

R:GGGTGACGGGCGGTATG

P:5′FAMATGGCCCTGAGGCAC3′Eclipse

12SrRNA??

??

F:GCTTCAATCCTCTATTTCATAA

R:GCTTCTTCCTTGAGTCTTA

P:5′FAMCCTCCTAGTCTTCATGCCAATCGT3′Eclipse

犮狔狋犫??

?

F:CTGCTGTCTTACTACTTCTA

R:GGTGTTTAGGTTTCGATC

P:5′FAMCACTTCCAGTCTTAGCAGCGG3′Eclipse

COⅠ??

?

F:TAATCGTCACCGCACATGCC

R:CTATGTCAGGAGCCCCAATTATCA

P:5′FAMACAAGCCAGTTCCCGAAGCCTCCA3′Eclipse

COⅠ??

?

F:ATCTACATGATTCATTACAATTAC

R:CTATGTTTTTGAGTTTTGAGTTCA

P:5′FAMATCTCAAACTACTCATACCCA3′TAMRA

ATP8??

?

F:TCCAGGTAAACCCTTCTTCCC

R:TTACGAGCAAGGGTTGATGGT

P:5′FAMATCACTTAGTCCAATAAGG3′MGBNFG

Dloop??

??

F:TGGAGCATCAGTAGACCTTACAATTT

R:GGCGGGAGGTTTTATATTGATAATAG

P:5′FAMCCCTGCACCTGGCCGGAGTC3′TAMRA

COⅠ??

?

F:AATCTTGCCTGGGTTTGGAA

R:CAGTAAATATGTGGTGGGCTCACA

P:5′FAMCATACTACTCCGGGAAAA3′MGBNFG

COⅠ??

犌犅/犜38164—2019

?1(?)

??????????

?

F:CCCTCCTCCTTTCATCCTCAT

R:GTCATAGCGGAACCGTGGATA

P:5′FAMCTATGAATCCGGGCCTC3′TAMRA

ND1??

??

F:GCCCTAACCCCTTAAGAAAAGAAT

R:AGTTGCTATGGCTAAGTCAAGTTTACAC

P:5′FAMCTTGCTTGAGCCACACC3′MGBNFG

12SrRNA??

?

F:CCGCCCAATCACCCAAA

R:GCCTCCGATTCATGTTAAGA

P:5′FAMTACTGAATYCTAGTAGCCAACCT3′MGBNFG

犮狔狋犫??

?:F??????;R??????;P????。

6??

??????,?????????。??????GB/T6682???????。

6.1????。

6.2???。

6.3????。

6.4??????(10mol

/L):?160mL??????80.0g????(NaOH),???,????

?,????????200mL。

6.5Na2EDTA??(500mmol/L,pH8.0):??18.6gNa2EDTA,??70mL????,????

???????(6.4),????????,?????,???????(6.4)?

pH

?8.0,?????

??100mL。?103.4kPa(121℃)?????20min。

6.6TrisHCl??(1mol/L,pH8.0):??121.1gTris???800mL????,?HCl?

pH

?

8.0,???????1000mL。?103.4kPa(121℃)?????20min。

6.7CTAB?????(pH8.0):??4.0gCTAB,16.364gNaCl,??20mL1mol/LTrisHCl?

?(pH8.0)(6.6),8mL500mmol/LNa2EDTA??(pH8.0),??70mL?????,????

200mL,?103.4kPa(121℃)?????20min。

6.8CTAB???:??1.0gCTAB,0.467gNaCl,??70mL?????,????200mL,?

103.4kPa(121℃)?????20min。

6.9???K(670U/mL):

??0.10g???33.5U/

mg

(Unit,????)????K??,??

5mL???,????,?????K????,????????-20℃。

6.10PCRMasterMix(2×),??????????PCR???。

7????

7.1????PCR?。

7.2???????????????。

7.3?????。

7.4???:

???≥12000犵。

犌犅/犜38164—2019

7.5pH?:0.01?。

7.6??:??0.1g、0.01g、0.001g。

8????

8.1?????

8.1.1??

????????,???????????,??????、?????????。

8.1.2????

???70%????????2?~3?,?????50mL??????????,-20℃?

???。

8.1.3???、

????

??????????50mL??????????,-20℃????。

8.2犇犖犃??

??GB/T35918?????。

?????DNA???????DNA。

8.3犇犖犃????????

??GB/T34796???????DNA???,??DNA???。

?:????PCR?????,??DNA?????5ng/μL~50ng/μL??。

8.4????犘犆犚??

???????????、?????????。

??????????????????????????,???????????????

?????????????,?????????。

???????2????????,???????????????,?Ct???????

??。????????25μL,??????2,??????3。

?2????犘犆犚??????

?????????/μL

PCRMasterMix(2×)1×12.5

????(10μmol/L)200nmol/L0.5

????(10μmol/L)200nmol/L0.5

??(10μmol/L)200nmol/L0.5

DNA??(5ng/μL~50ng/μL)—5.0

???—6.0

犌犅/犜38164—2019

?3????犘犆犚????

???????

???95℃10min;95℃15s,60℃1min,40???

??95℃10min;95℃15s,60℃1min,40???

??95℃10min;95℃15s,60℃1min,40???

??95℃10min;95℃15s,60℃1min,40???

??95℃10min;95℃15s,60℃1min,40???

???95℃10min;95℃15s,60℃1min,40???

??95℃10min;95℃15s,60℃1min,40???

??95℃10min;95℃15s,60℃1min,40???

??95℃10min;95℃15s,60℃1min,40???

??95℃10min;95℃15s,62℃1min,40???

?95℃10min;95℃15s,60℃1min,40???

?95℃10min;95℃15s,60℃1min,40???

??95℃10min;95℃15s,60℃1min,40???

??95℃10min;95℃15s,60℃1min,40???

??95℃5min;95℃15s,60℃40s,40???

?95℃5min;95℃15s,60℃40s,40???

?95℃5min;95℃15s,60℃40s,40???

?95℃10min;95℃15s,62℃40s,40???

?95℃2min;95℃15s,65℃80s,40???

?95℃2min;95℃15s,65℃80s,40???

??95℃2min;95℃15s,65℃80s,40???

?95℃10s;95℃5s,60℃30s,40???

??95℃10s;95℃5s,52℃10s,72℃34s,40???

?95℃10s;95℃5s,52℃10s,72℃34s,40???

9????

???????????,??????:

a)???:???????????,??????????,Ct?<30.0;

b)????:???????????,Ct??≥40.0;

c)????:???????????,Ct??≥40.0;

d)????:???????????,??????????,Ct?≤35.0。

10???????

10.1????

????9?????,???????????:

犌犅/犜38164—2019

a)?Ct?≤35.0,??????????,??????????A;

b)?Ct?≥40.0,??????????;

c)?35.0<Ct?<40.0,?????。??????Ct???<40.0,?????????;?

?????Ct?≥40.0,?????????。

10.2????

10.2.1??????,

???“??×××(????)??”。

10.2.2??????,

???“???×××(????)??”。

11??????????????

??GB/T27403—2008???D?????。

犌犅/犜38164—2019

??犃

(?????)

???????????????

???????????????(GenBank:MK028750.1):tggagtaatccttctgctcacagtaatagccacagcattt

ataggatacgtcctaccatgaggacaaatatcattctgaggagcaacagtca

???????????????(GenBank:GQ464309.1):gactaatattcgaaaatcccatccactaataaaaattgtaa

acaacgcattcattgaccttccagctccatcaaacatttcatcatggtgaaacttcggctccctcctaggag

???????????????(GenBank:MH880269.1):ttcattgatctccctgctccatcaaacatctcatcatgatg

aaactttggctctctcctaggcatctgcctaattctgcaaatcctcaccggcctattcc

???????????????(GenBank:MH110007.1):attctttgccttccacttcatcctgccatttattatcacggc

cctagtagccgtacacctattattcctacacgaaacaggctctaataacccgacaggaatctcctcagacatagacaaaatcccattccacccctactaca

caattaaagacatcctaggagcactgctactaatattaattctccttattctcgtactgttctcaccagacttattaggagatcctgacaactatactcccgc

taaccccctcaatacaccaccacacattaagccggaatgatatttcctattcgcatacgct

????????????????(GenBank:D14379.2):cctaccaccgaagcaatagtggacactgtagctactcaag

aagctttctcagaagttattgagagtgcacc

???????????????(GenBank:NC_039923.1):tagttatgtaaacaagactgttcgccagagtactaccgg

caatagcttaaaactcaaaggacttggcggtgctttatacccttctagaggagcctgttctataatcgataaaccccgataaacctcaccattccttgcta

ctacagtctat

???????????????(GenBank:GU220719.1):ctcccagtacgaaaggaccagagaaataaggccaacttt

aatcaagcgccttaaattaactaatgatttcatcttaattagatacacaaacatatcccgccc

???????????????(GenBank:LS992662.1):tggagtaatcctcctgctcgcgacaatggccacagcattca

taggctatgttttaccatgaggacaaatatcattttgaggggcaacagtcatcactaatcttctttcagcaatcc

???????????????(GenBank:KY786037.1):cggagtaatcctcctatttgcgacaatagccacagcattca

taggctacgtcttaccatgaggacaaatatcattctgaggagcaacagttattaccaacctcctttcagcaattccatatattggcacaagcctag

???????????????(GenBank:GQ240309.1):gagcaacccctcaacgtactaacagtaaccagacccaata

taattgaccaatggaccaagctaccccagggataacagcgcaatctcctccaagagtccatatcgacgaggaggt

??????????????(GenBank:EU914150.1):ggccacacaaatcctcacaggcctcctactggctatgcactac

accgcagacacatcccttgctttctcctcagtagccaacaca

??????????????(GenBank:AF363031):ccaccgcgaatcctcaatcaaccccactggggccactaccatcgc

agagcctggtcgatatttttcggttggggcgaccttggaggagaacaaatcctccaaaaacaaga

???????????????(GenBank:AM902516):ggatcactttgcccactttaagatggccctgaggcacgtaca

taccgcccgtcaccc

???????????????(GenBank:AB052720.1):gcttcaatcctctatttcataatcctcctagtcttcatgccaa

tcgtcagyattattgaaaataatctattaaaatgaagagtctttgtagtatatcaattacyttggtcttgtaaaccaaaaatggagaaccctatctccctaa

gactcaaggaagaagc

??????????????(GenBank:U20753.1):ctgctgtcttactacttctatcacttccagtcttagcagcgggaatc

actatattattaacagatcgaaacctaaacacc

??????????????(GenBank:NC_001913.1COX1):taatcgtcaccgcacatgcctttgtaataatcttctt

tatagtcatgcctattataattggaggcttcgggaactggcttgtccccctgataattggggctcctgacatag

??????????????(Genbank:KY964306.1):atctacatgattcattacaattacatcaataattataacattatt

tattttattccaactaaaaatctcaaactactcatacccagcaagcccagaatcaaccgaactcaaaactcaaaaacatag

犌犅/犜38164—2019

??????????????(Genbank:KY700649.1):tccaggtaaacccttcttccctcccctatgtacgtcgtgcatta

atggtttgccccatgcatataagcatgtacataatattatattcttacataggacatatcaactcaatctcacaattcattgatctgtcagcagtat

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