Listeria monocytogenes fluorescence real-time PCR kuaj

Real-time fluorescent PCR hauv vitro nrhiav pom Listeria monocytogenes.

Kev qhia tshwj xeeb: 96 T / lub thawv
Cov khoom siv tau khaws cia ntawm 2 - 8 ℃.
Lub sijhawm siv tau yog 12 lub hlis.

specification: 96T / lub thawv

Siv: Real-time fluorescent PCR hauv vitro nrhiav pom Listeria monocytogenes.

Txoj cai tshawb nrhiav: Cov khoom siv muaj Listeria monocytogenes tshwj xeeb primers. Thaum cov qauv muaj Listeria monocytogenes, Listeria monocytogenes DNA muab rho tawm tom qab ua ntej ua kom muaj txiaj ntsig tau nthuav dav los ntawm PCR. Thaum cov tshuaj tiv thaiv, Listeria monocytogenes tshwj xeeb fluorescent sojntsuam hybridizes nrog lub hom phiaj nucleic acid thiab yog tib lub sijhawm Taq enzyme Hydrolysis cais pawg fluorescent los ntawm pawg quenching ntawm qhov kev sojntsuam: kom muaj Listeria monocytogenes tuaj yeem txiav txim tau raws sijhawm. cov fluorescence increment.

Khoom thiab muaj pes tsawg leeg:

Muaj pes tsawg leeg (96T/khoom siv)	specification	kom muaj nuj nqis
Kev daws cov tshuaj DNA	3ml	1 raj
Listeria monocytogenes PCR tshuaj tiv thaiv	1100ul	1 raj
Kais enzyme (5U/ul)	20ul	1 raj
Listeria monocytogenes tswj qhov zoo	40ul	1 raj
Listeria monocytogenes Kev tswj tsis zoo	0.5ml	1 raj
phau ntawv qhia		1

Cov xwm txheej cia thiab hnub tas sij hawm ntawm cov khoom siv:
1. Cov khoom siv tau khaws cia ntawm 2 - 8 ℃.
2. Lub sijhawm siv tau yog 12 lub hlis.

Cov cuab yeej siv tau:
ABI7500, 9600 series, MJ opticon2, Bio-Rad thiab lwm yam fluorescent ntau qhov ntsuas PCR muaj

Txoj kev sim:
1. Siv 1ml ntawm cov tshuaj ua kom zoo rau hauv 1.5m1 cov menyuam tsis muaj menyuam centrifuge raj, centrifuge ntawm 10.000rpm rau 5min, thiab pov tseg cov supernatant; siv cov txheej txheem rho tawm phenol chloroform qub los kuaj cov ntawv rho tawm lossis khaws nws ntawm 20C kom txog thaum nws raug sim.
2. Muab PCR cov tshuaj tiv thaiv ntawm chav sov kom sib npaug, coj Taq enzyme tom qab yaj, thiab ntxiv Taq enzyme ntawm 2∪/raj, uas yog, txhua qhov kev sim tshuaj tiv thaiv kab mob tau npaj ua: PCR cov tshuaj tiv thaiv tshuaj 22.5u1+0.4ulTaq enzyme.
3. Ntxiv tus qauv: Thaw cov nucleic acid extract khaws cia ntawm -20 ° C ntawm chav sov thiab centrifuge ntawm 13,000 rpm rau 5 feeb. Qhov tsis zoo thiab kev tswj hwm tau yaj hauv chav sov ua ntej siv. Hauv txhua lub PCR cov tshuaj tiv thaiv raj, ntxiv 2 ul ntawm tus qauv lossis qhov tsis zoo thiab kev tswj hwm tau ua tiav hauv qib 1, npog lub raj, tso rau ntawm PCR ntsuas, thiab sau tus lej piv txwv.
4. On-board amplification thiab nrhiav chaw: qhov xwm txheej tshwm sim yog 95C: 3min, 1 lub voj voog; 95C: 5sec, 60C: 40sec (teeb liab tau txais), 40 mus. Cov tshuaj tiv thaiv kab mob tau teeb tsa rau 25u1.
Rau ntau-channel fluorescence PCR ntsuas, lub teeb liab tau teeb tsa rau Fam fluorescein.

Cov txiaj ntsig kev txiav txim:
Thov mus saib phau ntawv qhia kom ntxaws.

Ceev faj rau kev siv cov khoom siv:
1. Cov khoom siv no tsuas yog siv rau hauv vitro kuaj thiab tshawb fawb lub hom phiaj, thov nyeem tag nrho cov ntawv ntawm phau ntawv no ua tib zoo ua ntej pib siv nws.
2. Kev sim yuav tsum tau muab faib nruj me ntsis: thaj tsam npaj ua ntej PCR-npaj cov tshuaj reagents; cheeb tsam ua qauv-yuav tsum kuaj cov qauv, ua qauv; thaj tsam nrhiav pom-PCR kev nthuav dav, nrhiav pom.
3. Rau cov txheej txheem kev tswj hwm hauv chaw sim, thov saib mus rau qhov ua tiav ntawm cov qauv kev tswj hwm rau kev hloov pauv cov noob tso tawm los ntawm cov koom haum hauv tebchaws.

Yog tias koj xav paub ntau ntxiv, thov hu rau peb

Cov lus nug tsis tseem ceeb: Listeria monocytogenes

Hais txog Listeria

Listeria txhais

Listeria Nws yog ib qho facultative anaerobic kab mob uas yog tus neeg sawv cev ntawm listeriosis. Listeria yog ib tug kab mob Gram-positive uas yog ib tug tuab-walled phylum, muaj npe tom qab Joseph Lister. Nws tsuas yog siv cov zaub mov ua vector thiab yog ib qho ntawm cov khoom noj uas ua rau cov kab mob tuag taus.

Listeria yog ubiquitous nyob rau hauv ib puag ncig thiab muaj peev xwm pom nyob rau hauv feem ntau cov zaub mov. Nqaij, qe, nqaij qaib, nqaij nruab deg, khoom noj siv mis, zaub, thiab lwm yam tau raug pov thawj los ntawm Listeria kab mob. Listeria monocytogenes lom tuaj yeem ua rau cov ntshav thiab cov hlab hlwb. Ntau lub teb chaws tau ntsuas los tswj Listeria monocytogenes hauv cov zaub mov thiab tau teeb tsa cov qauv sib xws.

Listeria qhov chaw

Listeria tau pom thawj zaug hauv xyoo 1926 los ntawm tus kws tshawb fawb Askiv ntawm South African keeb kwm Murray hauv tus menyuam luav. Hauv kev ua koob tsheej nco txog leej txiv ntawm kev phais tshuaj tua kab mob niaj hnub no, tus kws kho mob Askiv Askiv Joseph Lister (1827-1912), xyoo 1940, nws tau hu ua Listeria monocytogenes ntawm 3rd International Congress of Microbiology.

Listeria hom

Lub thoob ntiaj teb lees paub Listeria monocytogenes muaj xya hom hauv qab no:
Listeria monocytogenes, Listeria mutans, Listeria innocolus, Listeria welsii, Listeria sileeri, Listeria monocytogenes, Listeria monocytogenes. Ntawm lawv, L. monocytogenes yog tib qho uas tuaj yeem ua rau tib neeg muaj kab mob. Nws yog cov kab mob ntawm cov kab mob zoonotic. Cov tshuaj lom neeg mob hnyav tuaj yeem ua rau cov ntshav thiab cov hlab hlwb. Listeria monocytogenes yog abbreviated li Listeria monocytogenes, uas yog ib tug kab mob Gram-zoo thiab belongs rau cov tuab-walled phylum.

Cov kab mob Listeria

Nyob rau hauv genus Listeria, tsuas yog Listeria monocytogenes tuaj yeem ua rau cov kab mob xws li mob meningitis, sepsis, nchuav menyuam, thiab kab mob neonatal rau tib neeg. Yog tias tus neeg tiv thaiv tsis muaj zog tiv thaiv kab mob Listeria, tus kab mob no loj heev thiab tus neeg tuag txog li 33%.

Cov tsos mob ntawm Listeria

Cov kab mob Listeria tuaj yeem nrog ua npaws, mob caj dab, ntuav lossis raws plab. Cov ntaub ntawv ntau dua tuaj yeem ua rau mob encephalitis thiab cov ntawv tshwj xeeb ntawm meningitis. Cov poj niam cev xeeb tub kuj muaj kev pheej hmoo ntawm kev cog lus Listeria. Cov poj niam cev xeeb tub yog 20 npaug ntau dua yuav kis Listeria dua li lwm tus. Txawm hais tias tsis muaj qhov nchuav menyuam lossis yug menyuam, tus menyuam mos tuaj yeem muaj tus kabmob kis no thaum yug los, yog li muaj kev tuag ntau heev.

Listeria monocytogenes

Dab tsi yog Listeria monocytogenes

Listeria monocytogenes yog cov kab mob ntawm cov kab mob zoonotic. Nws tuaj yeem ua rau listeriosis hauv tib neeg thiab tsiaj txhu. Tom qab kis tus kab mob, nws yog feem ntau tshwm sim raws li sepsis, meningitis thiab mononuclear hlwb. Vim hais tias cov kab mob yog ib qho kab mob hauv lub cev, kev tshem tawm los ntawm tus tswv tsev feem ntau yog nyob ntawm lub cev tiv thaiv kab mob. Yog li, listeriosis feem ntau yog nyob rau hauv neonates, cov neeg laus, thiab cov neeg uas muaj kev tiv thaiv kab mob. Listeria monocytogenes feem ntau kis tau los ntawm cov quav, thiab tuaj yeem nkag mus rau hauv lub cev los ntawm qhov muag thiab cov tawv nqaij puas thiab cov mucous membranes ua rau muaj kab mob.

Kev faib tawm ntawm Listeria monocytogenes

Listeria monocytogenes dav muaj nyob rau hauv cov xwm, tsis yooj yim rau khov thiab thawed, thiab tuaj yeem ua siab ntev osmotic siab. Cov kab mob no muaj nyob hauv av, dej saum npoo av, dej phwj tuaj, dej khib nyiab, nroj tsuag, silage, rotten zaub, yog li tsiaj tuaj yeem noj cov kab mob yooj yim.

Txoj kev sib kis ntawm Listeria monocytogenes

Listeria monocytogenes feem ntau siv cov khoom noj ua tus kab mob. Nws yog ib qho ntawm cov kab mob ua rau tuag taus ntau tshaj plaws, ua rau 20 mus rau 30% ntawm cov neeg mob tuag. Qhov kev tuag ntawm tus kab mob yog ntau dua li Salmonella thiab Botox. Cov neeg feem ntau noj cov cheese muag, cov nqaij qaib tsis muaj cua sov, cov dev tsis kub, cov mis nyuj tshiab, cov mis nyuj pasteurized, ice cream, steak nyoos, yaj chops, zaub qhwv zaub qhwv, celery, txiv lws suav, Fabkis pies, khov nqaij npuas nplaig thiab lwm yam. Kwv yees li 80% -90% ntawm cov neeg mob yog tshwm sim los ntawm cov zaub mov tsis huv.
Cov kab mob tuaj yeem nkag mus rau hauv lub cev los ntawm lub qhov muag thiab cov tawv nqaij puas thiab cov mucous membranes ua rau kis kab mob. Tom qab kis tus kab mob, cov poj niam cev xeeb tub kis tus me nyuam hauv plab los yog yug me nyuam los ntawm cov placenta los yog lub qhov dej yug me nyuam. Cov kab mob uas nyob hauv qhov chaw mos thiab lub ncauj tsev menyuam tuaj yeem ua rau muaj kab mob. Kev sib deev kuj yog ib txoj hauv kev ntawm kev sib kis ntawm tus kab mob, thiab muaj kev nce siab.

Listeria monocytogenes kab mob

Cov tsos mob ntawm Listeria monocytogenes

Txawm hais tias Listeria monocytogenes yuav mob tom qab nkag mus rau tib neeg lub cev yog cuam tshuam nrog kev ua phem ntawm cov kab mob thiab lub hnub nyoog thiab kev tiv thaiv kab mob ntawm tus tswv, vim hais tias cov kab mob no yog kab mob kab mob hauv lub cev, thiab kev tshem tawm los ntawm tus tswv tsev feem ntau yog nyob ntawm lub cev tiv thaiv kab mob. . Yog li ntawd, cov neeg raug tsim txom yog cov menyuam yug tshiab, cov poj niam cev xeeb tub thiab cov neeg laus dua 40 xyoo. Tsis tas li ntawd, cov neeg haus dej caw, cov neeg uas muaj lub cev tiv thaiv kab mob puas tsuaj los yog tsis zoo, cov neeg mob tau txais cov tshuaj tiv thaiv kab mob thiab corticosteroids, thiab kev hloov pauv hauv nruab nrog cev kuj raug rau cov kab mob. Cov tsos mob ntawm tus kab mob no, cov neeg laus noj qab nyob zoo muaj cov tsos mob me me xws li mob khaub thuas, menyuam mos, cev xeeb tub, cov poj niam cev xeeb tub, cov neeg mob tiv thaiv kab mob ua tsis taus pa, ntuav, hemorrhagic pob, suppurative conjunctivitis, kub taub hau, convulsions, coma, spontaneous rho menyuam Sepsis, meningitis, tuag.

Kev kho mob Listeria monocytogenes

Penicillin, ampicillin, gentamicin, erythromycin, thiab lwm yam tuaj yeem siv los kho cov kab mob Listeria monocytogenes. Erythromycin thiab lwm yam broad-spectrum tshuaj tua kab mob yog ua xua rau penicillin.

Listeria monocytogenes tiv thaiv

Listeria monocytogenes tuaj yeem muaj sia nyob ib txwm ua haujlwm thermal, kev kho cua sov tau tua cov kab mob sib tw hauv zej zog, ua rau Listeria monocytogenes yooj yim kom muaj sia nyob thaum tsis muaj kev sib tw ib puag ncig, yog li hauv kev ua zaub mov, cov tub ntxhais kub yuav tsum ncav cuag 70 ° C rau ntau tshaj 2 feeb. Listeria monocytogenes yog dav nyob rau hauv cov xwm, yog li txawm hais tias cov khoom tau overheated thiab ua tiav, Listeria monocytogenes yog tag nrho inactivated, tab sis nws yuav ua rau cov pa phem thib ob ntawm cov khoom, yog li tom qab ua noj ua haus, tiv thaiv cov pa phem theem nrab yog ib qho tseem ceeb heev. Vim Listeria monocytogenes tseem tuaj yeem loj hlob thiab rov tsim dua ntawm 4 ° C, cov khoom noj hauv lub tub yees tsis muaj cua sov ua rau muaj kev pheej hmoo ntawm cov zaub mov lom, thiab cov khoom noj hauv tub yees yuav tsum tau ua kom sov ua ntej noj.

Listeria monocytogenes kuaj

1. Kab lis kev cai kab lis kev cai

Txoj kev sim tshuaj rau Listeria hauv cov zaub mov yog ua kom muaj kev nplua nuj ua ntej lossis kev xaiv ua kom zoo, thiab siv cov kev sib cais thiab cov kab lis kev cai tsis txaus ntseeg rau kev sim tshuaj biochemical, kev sim hemolysis, thiab kev sib koom tes hemolysis (CAM P). Tom qab Listeria monocytogenes tau ntxiv serotyped. Enrichment thiab xaiv enrichment yog indispensable kauj ruam nyob rau hauv no txoj kev. Cov txheej txheem ntawm kev txhawb nqa feem ntau suav nrog: txoj kev ua kom txias txias thiab txoj kev cog qoob loo ib txwm muaj. Txoj kev ua kom txias txias yog kab lis kev cai ntawm 4 ° C rau 30 hnub, qee zaum mus txog ib xyoos. Nyob rau hauv chav tsev kub, cov kab mob yuav tsum tau coj mus rau 24h mus rau 7d. Yog li ntawd, txoj kev tshawb nrhiav ib txwm xav tau 7-11 hnub txhawm rau cais thiab txheeb xyuas Listeria monocytogenes, yog li cov txheej txheem tshawb pom ib txwm siv sijhawm ntev dua.

2. Serum agglutination

Raws li cov kab mob antigens thiab flagellar antigens, LM tau muab faib ua 16 serotypes: 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 5, 6a, 6b , 7. Antigen qauv tsis muaj dab tsi cuam tshuam nrog kev ua phem. Cov kab mob tseem ceeb rau tib neeg yog serotypes 1/2a, 1/2b, thiab 4b, suav txog li 90% ntawm cov neeg mob thoob ntiaj teb no.

3. Cov kev sim tshuaj molecular biology

3 .1 Kev soj ntsuam xyuas tshuab
Cov thev naus laus zis no yog qhov ntxov tshaj plaws ntawm cov kab mob molecular siv hauv kev tshawb nrhiav Listeria
Kawm technology. Thaum ntxov li 1988, Atin et al. cloned thiab sequenced ib 500bp DNA fragment ntawm Listeria beta-hemolysin noob, thiab synthesed plaub oligonucleotide probes los ntawm qhov sib lawv liag, sau lub sojntsuam nrog 32P, thiab ua ib tug dot hybridization xyaum sim Nws tau lees tias qhov kev sojntsuam tsuas reacted nrog Listeria monocytogenes. yog qhov tsis zoo nrog lwm hom Listeria thiab cov kab mob uas tsis yog-genus, qhia qhov tshwj xeeb zoo.

3.2 Polymerase chain reaction (PCR) nrhiav tshuab
Txoj kev loj hlob ntawm PCR yog qhov paub tab, thiab nws tau siv dav hauv kev tshawb nrhiav Listeria. PCR amplification tshwj xeeb primers yog tsim los raws li qhov tshwj xeeb virulence gene sequence of Listeria monocytogenes, thiab ib qho yog tsim los ntawm cov kab ke tshwj xeeb hauv genome ntawm Listeria monocytogenes. Feem ntau siv cov hom phiaj ua ntu zus yog cov kab mob virulence xws li hly A, iap, inl, Dth-18. Qhov kom zoo dua ntawm kev siv PCR txoj kev los kuaj Listeria yog qhov tshwj xeeb ntawm txoj kev, tab sis qhov tsis zoo yog qhov tsis tshua muaj siab; cov qauv kab lis kev cai yog amplified tom qab tshuaj extraction, uas txhim kho tus nqi ntawm cov qauv; thiab tuaj yeem "nyob, tsis tuaj yeem cog tau" Listeria monocytogenes tsis tuaj yeem kuaj pom los ntawm cov txheej txheem kev nplua nuj ib txwm muaj.