- Khong co dap Un gm iln djch di truyen
2. PHl/ONG PHAP CHAN DOAN BENH DON BAO K* SINH
2.5. Cac phuang phap chan doan mien djch benh dan bao dudng mau
* Phuang phap midn dich ELISA (Enzym Linked Immunosorbent Assay)
Phuang phap ELISA dimg de phat hien khang nguyen (cdn goi la ELISA khang nguyen) da dugc Nantulya va Lindquist md ta nam 1989. Cac bao cao ve ket qua sir
dung phuang phap nay cho tha'y, ky thuat ELISA nhay han so vdi cac phuang phap giam dinh khac va ra't dac hieu vdi cac loai Trypanosoma sp..
Phuang phap ELISA gian tiep su dung dia nhua duac gin khang nguyen da bifift. Khang the nghi co trong huyet thanh con vat nghi mac benh se ket hap vdi khang nguydn da duac gan vao dia. Sau do, mot khang thd khang lai IgG cua bo duac gan vdi men Peroxydase duoc nho vao se ket hap vdi khang the ne'u co trong huye't thanh cdn x6t nghiem. Su cd mat cua men Peroxydase dugc phat hien nhd mdt ca chat (Substrate) vdi su xua't hien mau. Tuy theo chat lam hien mau (Chromogene) ma khi doc k£t qua se su dung cac kinh dgc khac nhau.
Phuang phap ELISA gdm cac budc sau:
Budc 1: gan khang nguyen vao dia nhua, ii dia d 37(IC trong 2 gid hoac u qua dem 6 4°C.
Budc 2: rua dia 3 ian bang dung dich PBS (Phosphat Buffered Saline). Budc 3: nhd huyet thanh nghi. U dia d 37°C trong 1 gid
Budc 4: rira dia 3 ian bang dung dich PBS
A %
Budc 5: nhd IgG cua thd hoac de khang IgG cua bd gan vdi men Peroxydase. U dia d 37°C trong 1 gid.
Budc 6: rira dia 3 ian bang dung dich PBS. Budc 7: nhd ca chat
Budc 8: doc ket qua bang quang pho ke (Spectrophotometer)
Do nhay cua phuang phap ELISA phat hien khang the dat den 9 0 -9 5 % va dugc hau het cac tac gia thira nhan.
* Cac phuang phap huye't thanh hgc
Mot vai ky thuat phat hien khang the da dugc khao nghiem de chan doan benh do ky sinh triing dudng mau gay ra d dong vat, bao gom: phuang phap ELISA tren dia nhua (Luckins, 1977) va phuang phap ngung ke't tren card (thudng dugc ggi la phuang phap CARD TEST). Nhin chung, cac phuang phap nay cd do nhay cao nhung tinh dac hieu thap. Doi khi, cac phuang phap nay cd phan ung cheo nen khdng the phan biet cac loai vdi nhau. Ngoai ra, phuang phap huyet thanh hoc cd the xac dinh duac con vat mac benh nhung chua chac da xac dinh dugc tinh trang benh luc chan doan (vi cd the luc chan doan thi con vat da khoi benh nhung khang the van cdn trong huyet thanh). Trong cac phuang phap huyet thanh de chan doan benh dan bao dudng mau gay ra thi phuang phap khang the huynh quang gian tiep (IFAT: Indirect Fluorescent Antibody Test) duac coi la phuang phap thich hgp nhat de phat hien khang the’ dac hieu cua loai (species/specific antibody).
Phuang phap cua Wilson (1969) da dupe Katende va cs (1987) cai tien bucfc chuain bi khang nguyen, do la co dinh don bao tuoi len tieu ban bang h6n hop Aceton lanh 80% va Formalin 0,25%.
Theo Plant va cs (1976), Well va cs (1982), cac buoc tien hanh nhu sau:
Buoc 1: chuan bi tieu ban dan mau cua con vat mac benh ky sinh trung duang mau nang (hoac tieu ban tu huydn dich chua ky sinh trung). De kho trong didu kien nhiet dp moi truang roi co dinh bang c6n Aceton (hoac h6n hop Aceton lanh 80% va Formalin 0,25%), de trong 5 phut.
Buoc 2: dung but chi m a ve cac vong tron co duang kmh 5 mm a cac viing khac nhau trong vung dan cua tieu ban.
Buoc 3: dung pipet hut cac huyet thanh nghi da pha loang 1/40 nho rieng re vao cac vong tron vira khoanh tren tieu ban. Chu y m6i vong tron deu dupe phu km huyet thanh.
Budc 4: u h6n hop khang nguyen - huyet thanh nghi 30 phut trong tu am 37°C.
Buoc 5: rua tieu ban 3 lan bang dung dich PBS, m6i lan 5 phut a 4llC co lac nhe. Dd kho tieu ban a nhiet dp phong.
Buoc 6: nho khang the IgG cua tho hoac de khang IgG cua bo gan vai thuoc nhuom huynh quang - fluorescent isothiocyanate.
Buoc 7: u va rira tieu ban nhu tren, trang bang nuac cat. Dd kho tieu ban a nhiet do phong.
Buoc 8: gdn lamen vai dung dich PBS hoac Glycerin va kiem tra bang kmh hidn vi huynh quang.
* Phuang phap phat hien ADN cua dan bao bang phan ung PCR (Polymerase Cham Reaction).
PCR la phuang phap hien dai nhat, mai duac dua vao ung dung de chan doan mot vai benh ky sinh trung d uang mau.
Nguydn ly cua phan ung PCR la dua vao phan ung chu6i Polymerase de’ xac dinh su co mat ADN ciia ky sinh trung ducmg mau a dong vat.
Phuang phap PCR co do nhay va do chfnh xac rat cao. song phuang phap nav doi hoi phai co trang thiet bi hien dai va ky thuat cao. Hien nay. phuang phap PCR m ai duac thu nghiem a mot so phong thi nghiem hien dai.
TAI LI$U THAM KHAO CHl/ONG 6 * Tieng Viet
1. Drozdz J. va Malczewski (1971), Noi ky sinh vat va benh ky sinh vat cua gia sue a Viet Nam, Nha xua't ban khoa hoc va ky thuat, Ha N6i, Tr. 10 - 19.