Radionuclides in Molecular Technology for Diagnosis of Communicable Diseases
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IAEA-TECDOC-748 Radionuclides in molecular technology for diagnosis of communicable diseases Edited by Professor Savanat Tharavanij and Dr. Srisin Khusmith INTERNATIONAL ATOMIC ENERGY AGENCA / Y The IAEA does not normally maintain stocks of reports in this series. However, microfiche copies of these reports can be obtained from ClearinghousS I N I e International Atomic Energy Agency Wagramerstrasse 5 0 10 P.Ox Bo . A-1400 Vienna, Austria Orders should be accompanied by prepayment of Austrian Schillings 100,- in the form of a cheque or in the form of IAEA microfiche service coupons which may be ordered separately from the INIS Clearinghouse. originatine Th g Sectio f thino s documen IAEe th An i t was: Nuclear Medicine Section International Atomic Energy Agency Wagramerstrasse 5 P.O. Box 100 A-1400 Vienna, Austria RADIONUCLIDES IN MOLECULAR TECHNOLOGY FOR DIAGNOSI COMMUNICABLF SO E DISEASES IAEA, VIENNA, 1994 IAEA-TECDOC-748 ISSN 1011-4289 Printe IAEe th AustriAn y i d b a May 1994 FOREWORD Advance moleculan si r techniques continu madevee n b a t ro e a t increasin g rate. Thes related ean d procedures mann ,i f whicyo h radionuclide tracers pla importann ya t role, have f immensproveo e b o nt e potentia t onl no researchlyn i t als routinbu ,n o i e diagnosis of communicable diseases. Clinical problems, both diagnostic and therapeutic, are being solve usiny db approachew gne s made possibl advene th y f theseb o t e techniquesn I . diagnosis the amplification of DMA by the polymerase chain reaction and hybridization with P-labelle32 d DMA probes has excited clinical microbiologists for such methods can specifically identify organisms directl clinican i y l specimen reducn ca e timd eth ean s required to identify fastidious pathogens, even in diseases such as tuberculous meningitis and Chagas disease, in which the paucity of the infectious organisms had previously frustrated laboratory diagnosi conventionay sb l techniques thest Ye . e technique oftee sar n inaccessible to many biomedical scientists and clinicians in developing countries and there is a clear need to add these powerful techniques to the range of tools available in developing countrie r researcsfo diagnosid han communicabln si e diseases. One of the roles of international organizations is to assist in the transfer of such new technologie developino st g countries. With this objective IAEe th , A organizee th t da Facult f Tropicayo l Medicine, Mahidol University, Bangkok trainin,a g cours Septembeen i r 1992 on "Recent Nuclear Techniques in Diagnosis of Communicable Diseases" which was attende participant6 1 y db s fro Membe1 m1 r State Asie Pacifid th aan f so c Regiond an , a semina Novemben i r r 199 "Immunoassan 2o Labelled y an Probe A dDN Diagnosin si f so Communicable Diseases" whic attendes participanth2 wa 11 y db s fro Membem7 1 r States. The course focused largely on the teaching of molecular techniques, whereas the seminar presented the experiences of various experts in the application of such techniques in research and diagnosis. This TECDOC contains those aspects of the proceedings from the two events which focus on molecular techniques, and is in two parts: Part I contains the laboratory protocols that were taught at the course and Part II contains 9 of the 43 papers presente seminae th t da r which illustrat applicatioe eth f moleculano r techniques. EDITORIAL NOTE preparingIn this document press,for IAEAthe staffof have pages madethe up from the original manuscripts as submitted by the authors. The views expressed do not necessarily reflect those of the governments of the nominating Member States or of the nominating organizations. The use of particular designations of countries or territories does not imply any judgement by publisher,the legalthe IAEA, to status the as of such countries territories,or of their authoritiesand institutions delimitationthe of or of their boundaries. The mention of names of specific companies or products (whether or not indicated as registered) does implyintentionnot any infringeto proprietary rights, should construednor be it an as endorsement or recommendation on the part of the IAEA. The authors are responsible for having obtained the necessary permission for the IAEA to reproduce, translate or use material from sources already protected by copyrights. CONTENTS Introduction ................................................... 7 . I LABORATORY PROTOCOL MOLECULAR SFO R TECHNIQUES Radioiodinatio f proteinno solution si n ...............................1 1 . J.B. Castelino Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting for protein antigen analysis ................................ 13 Pramuan Tapchaisri, Srisin Khusmith, Yuwaporn Ruangkunaporn Selected techniques in recombinant DNA technology ...................... 21 Pramuan Tapchaisri Detection of enteroinvasive Escherichia col! on a colony blot filter paper by hybridization with a 17 kb probe .................................. 31 Oralak Serichantalergs, Channarong Sanghiran, Wipawee Usawatanakul, Korbkit Cherdchu Pathogen detection by the polymerase chain reaction ..................... 41 Suwicha Tim Chitpatima, D.R. Dvorak, Dhana Settachan, Jaturaporn Pornsilpatip, Unchalee Visawapoka II. APPLICATIONS OF MOLECULAR TECHNIQUES In vitro application radionuclidef so communicabln si e disease overvien A s- w ...9 .6 /. Nath Detection and identification of infectious organisms: Application of molecular probe technology ....................................5 7 . S.F. Yap Application of the polymerase chain reaction and molecular probe technology for the diagnosis of tuberculosis .................................. 79 S.F. Yap, Y.C. Chert, P.W. Wong, T.S. Soo-Hoo Detectio f Mycobacteriumno tuberculosis clinican i l samples usine gth polymerase chain reaction: Avoiding amplicon contamination ............3 8 . A. Kolk, L. Kox, Dhanida Rienthong, A. Medo Miranda, Nibondh Udomsantisuk, K. Ellis, J. van Leeuwen, S. Kuijper A frequencstude th f yo f infectioyo f peripherano l blood mononuclear cellf so chronic hepatitis B virus carriers using the polymerase chain reaction and hybridization analysis .......................................... 93 S.F. Yap, P.W. Wong, K.L. Goh, N.W. Wong Expression-PCR rapiA : d metho vitrn i r od fo expressio productR PC f nso .......7 9 . K.C. Kain, P.A. Orlandi, D.E. Lanar A comparative study of using DNA probe (pPF 14) and microscopy in diagnosis of Falciparum malaria .........................................3 10 . Jianliang Yang, Yongqi Kong, Cunxing Yang, Huiming Lu Application of molecular probes in histopathology of communicable diseases .... 107 L.M. Loo/, P.L. Cheah Epstein-Barr Virus (EBV) gene expressio HIV-associaten i d oral hairy leukoplaki5 11 . a R. Pathmanathan, K. Gilligan, L Resnick, N. Raab-Traub List of Partipants .............................................. 121 INTRODUCTION Molecular techniques continue to advance at a striking and ever increasing pace and makinw areno g significant inroad mann si y clinicae areath f so l microbiology laboratory. These include method r straisfo n typing suc plasmis ha d A fingeDN f ro printinge us e th , hybridization tests for identification of pathogens, and the use of polymerase chain reaction deteco t t pathogens presen vern numberi tw ylo clinican si l specimens consequencA . e of this phenomenal rat f progreseo thesn si e method bees sha n that many cliniciand san biomedical scientists in developing countries have found it impossible to keep pace with current developments, a situation exacerbated by the free use of jargon, and, as with all rapidly growing fields, it will be some time before comprehensive textbooks catch up. This technical document has been prepared with the view to filling the resultant vacuum. It is based on a training course and a seminar organized jointly by the International Atomic Energy Agency and Mahidol University, Bangkok, during 1992. In laboratories dealing with diagnosis of communicable diseases, the first analysis of clinical materia usualls i l y don lighy eb t microscopy after suitable staining. Whilst helpful in guiding therap r acutelyfo l patientsyil , microscopy techniques lac botn ki h sensitivity and specificity. Thus cultures remai gole nth d standard. However cleas i t ,i r tha mann i t y case gole sth d standar 100a t %no d s sensitivei . Moreover, culture method generan i e sar l slo laboriousd wan t therincreasen a Ye . es i d desire from physician havo st e results more rapidly. The need for rapid results is partially filled by immunological tests such as radioimmunoassays, ELISA (enzyme linked immuno-sorbent assay) and Western blot. The las thesf to e method labelline th indicato e d th sf an g o r reagent with 125iodin describee ear d in the first two papers of this TECDOC. These immunological tests, though sensitive, sometimes lack specificity, particularl developinn yi g countries where tests categorizes da 'highly specific industrializen i ' d countries cross-react wit hose hf th microorganism o t s present in many developing countries. The second forma r rapifo t d testin nucleigs i c acid hybridization earls A 198.s ya 0 it was demonstrated that genetic information contained within an organism could be exploited as a means for its identification. Enterotoxigenic Escherichia coli were identified in stool samples after growth on agar plates. Since then there have been numerous reports on the effectiveness of DNA probes for identification of pathogenic microorganisms ranging from viruse helminthio