Listeria monocytogenes fluorescence lub sijhawm PCR kuaj

Real-time fluorescent PCR hauv vitro kuaj 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 kuaj 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 kev txhim kho tau nthuav dav los ntawm PCR. Thaum lub sijhawm tshuaj tiv thaiv, Listeria monocytogenes-tshwj xeeb fluorescent sojntsuam sib xyaw nrog lub hom phiaj nucleic acid thiab ib txhij Taq enzyme Hydrolysis txhawm rau cais cov pab pawg fluorescent los ntawm pab pawg quenching ntawm kev sojntsuam: yog li muaj Listeria monocytogenes tuaj yeem txiav txim hauv lub sijhawm tiag tiag qhov nce ntawm fluorescence.

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 cov tshuaj tiv thaiv	1100ul	1 raj
Kais enzyme (5U/ul)	20ul	1 raj
Listeria monocytogenes tswj tau zoo	40ul	1 raj
Listeria monocytogenes Tsis Zoo Tswj	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

Txog listeria

Listeria txhais

Listeria yog cov kab mob hu ua anaerobic uas yog tus neeg sawv cev ntawm listeriosis. Listeria yog cov kab mob Gram-positive uas yog rau cov phab ntsa tuab, muaj npe tom qab Joseph Lister. Nws feem ntau yog siv zaub mov raws li vector thiab yog ib qho ntawm cov kab mob ua rau tuag tau ntau tshaj plaws.

Listeria muaj nyob thoob plaws ib puag ncig thiab tuaj yeem pom muaj nyob hauv cov khoom noj feem ntau. Nqaij, qe, nqaij qaib, nqaij nruab deg, khoom siv mis nyuj, zaub, thiab lwm yam tau raug pov thawj los ua qhov kis mob Listeria. Listeria monocytogenes lom tuaj yeem ua rau mob ntshav thiab mob hlwb. Ntau lub tebchaws tau ntsuas ntsuas los tswj Listeria monocytogenes hauv zaub mov thiab tau teeb tsa cov qauv sib xws.

Listeria qhov chaw

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

Listeria hom

Thoob ntiaj teb lees paub Listeria monocytogenes muaj xya yam kab mob hauv qab no:
Listeria monocytogenes, Listeria mutans, Listeria innocolus, Listeria welsii, Listeria sileeri, Listeria monocytogenes, Listeria monocytogenes. Ntawm lawv, L. monocytogenes tsuas yog ib qho uas tuaj yeem ua rau tib neeg muaj kab mob. Nws yog kab mob ntawm cov kab mob zoonotic. Kev ua tshuaj lom hnyav tuaj yeem ua rau cov ntshav thiab cov ntaub so ntswg puas. Listeria monocytogenes yog sau ua Listeria monocytogenes, uas yog kab mob Gram-positive thiab yog rau cov phylum-walled tuab.

Kev kis tus kab mob Listeria

Hauv genus Listeria, tsuas yog Listeria monocytogenes tuaj yeem ua rau muaj kab mob xws li mob meningitis, sepsis, nchuav menyuam, thiab kev yug menyuam hauv menyuam yaus. Yog tus neeg tsis muaj kev tiv thaiv kab mob txhim kho Listeria, tus kab mob no loj heev thiab cov neeg tuag nce txog 33%.

Cov tsos mob Listeria

Kev kis tus kab mob Listeria tuaj yeem nrog ua npaws, mob caj dab, ntuav lossis raws plab. Cov ntaub ntawv nyuaj dua tuaj yeem ua rau mob hlwb thiab qee hom mob meningitis. Cov poj niam cev xeeb tub kuj tseem pheej pheej kis Listeria. Cov poj niam cev xeeb tub muaj 20 zaus yuav kis tau Listeria ntau dua li lwm tus. Txawm hais tias tsis muaj kev nchuav menyuam lossis tseem yug menyuam, tus menyuam yug tshiab tuaj yeem muaj tus kabmob sib kis no txij thaum yug los, yog li muaj cov neeg tuag coob heev.

Listeria monocytogenes

Listeria monocytogenes yog dab tsi

Listeria monocytogenes yog kab mob ntawm cov kab mob zoonotic. Nws tuaj yeem ua rau listeriosis hauv tib neeg thiab tsiaj. Tom qab kis tus kab mob, nws feem ntau tshwm sim li sepsis, meningitis thiab mononuclear cells. Vim tias cov kab mob yog kab mob hauv lub cev, nws tshem tawm los ntawm tus tswv tsev feem ntau yog nyob ntawm kev ua haujlwm ntawm lub cev tsis muaj zog. Yog li ntawd, listeriosis feem ntau yog rau cov menyuam mos, cov neeg laus, thiab cov neeg tsis muaj kev tiv thaiv kab mob. Listeria monocytogenes feem ntau kis los ntawm cov quav, thiab tseem tuaj yeem nkag mus rau hauv lub cev los ntawm lub qhov muag thiab ua rau tawv nqaij puas thiab ua rau cov hnoos qeev kom ua rau kis mob.

Kev faib tawm ntawm Listeria monocytogenes

Listeria monocytogenes dav muaj nyob rau hauv qhov, tsis yooj yim kom khov thiab yaj, thiab tuaj yeem zam siab osmotic siab. Cov kab mob no muaj nyob hauv av, dej nto, dej phwj tuaj, dej tsis huv, nroj tsuag, ntsaum, zaub lwj, yog li cov tsiaj tuaj yeem noj tau cov kab mob yooj yim.

Txoj kev sib kis ntawm Listeria monocytogenes

Listeria monocytogenes tsuas yog siv zaub mov ua tus neeg kis mob. Nws yog ib qho ntawm cov kab mob txaus ntshai tshaj plaws los ntawm cov zaub mov, ua rau 20 txog 30% ntawm cov neeg muaj mob tuag. Cov neeg tuag tseem siab dua li Salmonella thiab Botox. Cov neeg feem ntau noj cov cheese mos, cov nqaij qaib tsis sov, tsis kub cev, mis nyuj tshiab, mis nyuj pasteurized, mis nyuj khov, cov nqaij nyoos nyoos, cov nqaij yaj, zaub qhwv zaub qhwv, zaub qhwv, zaub xas lav, txiv lws suav, Fabkis kib, npliag nqaij npua khov thiab lwm yam. Kwv yees li 80% -90% ntawm cov xwm txheej tshwm sim los ntawm cov zaub mov tsis huv.
Cov kab mob tuaj yeem nkag mus rau hauv lub cev los ntawm ob lub qhov muag thiab ua rau cov tawv nqaij puas thiab cov mucous membrane ua rau kis mob. Tom qab kis tus kab mob, cov poj niam cev xeeb tub kis tus menyuam hauv plab lossis tus menyuam yug tshiab los ntawm lub tsho me nyuam lossis lub qhov dej yug menyuam. Cov kab mob uas nyob hauv qhov chaw mos thiab lub ncauj tsev menyuam kuj tuaj yeem ua rau kis mob. Kev sib deev los kuj tseem yog txoj hauv kev tau kis tus kab mob, thiab muaj qhov nce zuj zus.

Listeria monocytogenes kab mob

Listeria monocytogenes cov tsos mob

Txawm hais tias Listeria monocytogenes dhau los ua mob tom qab nkag mus rau tib neeg lub cev cuam tshuam nrog virulence ntawm cov kab mob thiab hnub nyoog thiab kev tiv thaiv kab mob ntawm tus tswv tsev, vim hais tias cov kab mob yog kab mob hauv cov kab mob parasitic, thiab nws tshem tawm los ntawm tus tswv feem ntau yog nyob ntawm kev ua haujlwm ntawm lub cev tsis muaj zog . Yog li ntawd, cov neeg raug mob yog cov menyuam yug tshiab, poj niam cev xeeb tub thiab cov neeg laus dua 40 xyoo. Tsis tas li ntawd, kev quav dej quav cawv, cov neeg muaj lub cev tsis muaj zog lossis lub cev tsis muaj zog, cov neeg mob tau txais cov tshuaj tiv thaiv kab mob tiv thaiv kab mob thiab corticosteroids, thiab kev hloov pauv hauv lub cev kuj tseem kis tau tus kab mob. Cov tsos mob ntawm tus kab mob, cov neeg laus noj qab haus huv muaj cov tsos mob zoo li mob khaub thuas, cev xeeb tub, poj niam cev xeeb tub, cov neeg tsis muaj kev tiv thaiv kab mob ua tsis taus pa, ntuav, ntuav ntshav, ua rau mob sib txuas, kub cev, ntuav, tsis nco qab, rho menyuam tawm, mob qog noj ntshav, Sepsis txog thaum tuag.

Listeria monocytogenes kho

Penicillin, ampicillin, gentamicin, erythromycin, thiab lwm yam tuaj yeem siv rau kev kho mob Listeria monocytogenes. Erythromycin thiab lwm yam tshuaj tua kab mob dav dav ua rau tsis haum rau penicillin.

Listeria monocytogenes tiv thaiv

Listeria monocytogenes tuaj yeem muaj sia nyob ib txwm ua cov cua sov, 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 ib puag ncig, yog li hauv kev ua zaub mov, qhov kub thiab txias yuav tsum ncav cuag 70 ° C ntau dua 2 feeb. Listeria monocytogenes tau nthuav dav nyob rau hauv xwm, yog li txawm tias cov khoom tau dhau los ua kom sov thiab ua tiav, Listeria monocytogenes tau ua tsis tiav, tab sis nws yuav ua rau muaj kuab paug theem ob ntawm cov khoom, yog li tom qab ua noj, tiv thaiv kev ua qias tuaj thib ob yog qhov tseem ceeb heev. Vim tias Listeria monocytogenes tseem tuaj yeem loj hlob thiab rov tsim dua ntawm 4 ° C, cov khoom noj hauv lub tub yees tsis sov ua rau muaj kev pheej hmoo ntawm zaub mov lom, thiab cov khoom noj txias yuav tsum tau muab rhaub ua ntej noj.

Listeria monocytogenes kuaj

1. Kab mob kab lis kev cai

Txoj kev sim ib txwm rau Listeria hauv zaub mov yog ua kom tiav ua ntej lossis xaiv cov khoom ntxiv, thiab siv cov cais thiab kev coj noj coj ua uas tsis txaus ntseeg rau kev sim tshuaj lom biochemical, kev sim hemolysis, thiab kev koom tes hemolysis thwmsim (CAM P). Tom qab Listeria monocytogenes tau txuas ntxiv. Kev txhim kho thiab xaiv cov kev txhawb ntxiv yog cov kauj ruam tseem ceeb hauv txoj kev no. Cov txheej txheem ntawm kev txhawb nqa feem ntau suav nrog: txheej txheem ua kom txias thiab txheej txheem kev cog qoob loo ib txwm muaj. Txoj kev ua kom txias txias yog cog ntawm 4 ° C rau 30 hnub, qee zaum txawm tias mus txog ib xyoos. Hauv chav sov, cov kab mob yuav tsum tau cog rau 24h txog 7d. Yog li, txoj kev kuaj pom ib txwm xav tau 7-11 hnub txhawm rau cais tawm thiab txheeb xyuas Listeria monocytogenes, yog li cov txheej txheem ib txwm tshawb pom lub sijhawm ntev dua.

2. Ntshav agglutination

Raws li kab mob antigens thiab flagellar antigens, LM tau muab faib ua 16 hom kab mob: 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 ua rau virulence. Cov kab mob tseem ceeb rau tib neeg yog serotypes 1/2a, 1/2b, thiab 4b, suav txog kwv yees li 90% ntawm cov neeg mob thoob ntiaj teb ntawm tus kabmob no.

3. Molecular biology xeem txoj kev

3 .1 Kev sojntsuam thev naus laus zis
Cov thev naus laus zis no yog lub cev txheej txheem molecular ntxov tshaj plaws uas siv hauv kev tshawb nrhiav Listeria
Kawm paub siv tshuab. Thaum ntxov li 1988, Atin li al. cloned thiab ua ntu zus 500bp DNA tawg ntawm Listeria beta-hemolysin noob, thiab sib sau ua ke plaub oligonucleotide sojntsuam los ntawm qhov xwm txheej no, sau lo lus sojntsuam nrog 32P, thiab ua qhov kev sib tw dot hybridization tsis zoo nrog lwm hom Listeria thiab cov tsis muaj kab mob, qhia qhov tshwj xeeb zoo.

3.2 Polymerase chain reaction (PCR) nrhiav tshuab
Kev txhim kho PCR yog qhov paub tab heev, thiab nws tau siv dav hauv kev tshawb nrhiav Listeria. PCR kev nthuav dav tshwj xeeb cov txheej txheem tau tsim los raws qhov tshwj xeeb virulence gene sequence of Listeria monocytogenes, thiab ib qho yog tsim los raws cov ntu tshwj xeeb hauv genome ntawm Listeria monocytogenes. Cov hom phiaj siv feem ntau yog cov virulence genes xws li hly A, iap, inl, Dth-18. Qhov zoo ntawm kev siv PCR txoj hauv kev txhawm rau txheeb xyuas Listeria yog qhov tshwj xeeb ntawm txoj hauv kev, tab sis qhov tsis zoo yog qhov tsis nkag siab; cov qauv kev coj noj coj ua tau nthuav dav tom qab rho tawm cov tshuaj, uas txhim kho qhov ntsuas pom ntawm tus qauv; thiab tuaj yeem A "nyob, tsis tuaj yeem cog" Listeria monocytogenes tsis tuaj yeem kuaj pom los ntawm cov txheej txheem kev txhim kho ib txwm muaj.