Molecular techniques

Molecular techniques, other than PCR

1. Molecular Diagnosis Dr.M.Malathi Postgraduate II year Department of Microbiology Chengalpattu medical college

2. Molecular technology Molecular diagnosis is the most appropriate for infectious agents that are difficult to detect, identify, or test for susceptibility in a timely fashion with conventional mehtods . Need of molecular methods is most important in diagnosis of Mycobacterium tuberculosis, Chlamydia trachomatis , meningoencephalitis syndrome and respiratory viral illness.

3. Where traditional methods fails?? Microscopy gives false positive results in T.vaginalis , N.gonorrhoeae Intracellular pathogens – Chlamydia Subtyping in case of HSV, HPV, HCV Microbial growth is slow – Myco.tb

4. How it works?? Every organism contains some unique, species specific DNA sequences Molecular diagnostics makes the species specific DNA visible

5. Applications Classification of organism based on genetic relatedness (genotyping) Identification and confirmation of isolate obtained from culture Early detection of pathogens in clinical specimen Rapid detection of antibiotic resistance Detection of mutations

6. Differentiation of toxigenic from non toxigenic strains Detection of microorganisms that lose viability during transport, impossible, dangerous and costly to culture, grow slowly or present in extremely small numbers in clinical specimen Apart from microbiology, useful in forensic medicine

7. Techniques used Nucleic acid hybridisation Amplification techniques Plasmid profiling Nucleotide sequencing Restriction Fragment Length Polymorphism Pulse Field Gel electrophoresis

8. TARGET AMPLIFICATION PCR Based Non PCR based (Isothermal)  NASBA  TBA  SDA  LAMP

9. Nucleic Acid Sequence Based Amplification Isothermic non PCR procedure Definition: a primer dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature ( 1991, J Compton) 3 SR : self sustained sequence replication 3 enzymes : AMV reverse transcriptase, Ribonuclease H, T7 RNA polymerase

10. Immediately after the invention of NASBA, it was used for the rapid diagnosis and quantification of HIV-1 in patients` sera Quicker than PCR Isothermal More sensitive Used to detect pathogenic viruses with ssRNA genomes Eg : influenza A, Foot and mouth disease virus, SARS, HboV , Trypanosoma brucei

11. NASBA PCR RNAase H is the denaturing agent Heat is the denaturing agent Isothermal 41deg C- no need of thermocycler Thermal variation – thermocycler needed For ssRNA Both DNA and RNA

12. Transcription B ased Amplification Useful in the amplification of ss RNA rather than DNA Similar to NASBA Developed by Gen-probe, Inc Used in clinical laboratories to detect Chlamydia trachomatis and Neisseria gonorrheae from clinical specimens

13. Strand Displacement Amplification Isothermal Based on restriction endonuclease nicking its recognition site and a polymerase extending the nick at its 3` end displacing the downstream strand. Required restriction enzyme cleavage of the DNA sample prior to amplification

14. Normally restriction enzyme cleavage produces dsDNA , which is not suitable template for SDA By incorporating alpha thio substituted nucleotides , a double stranded hemiphosphorothioated DNA is created where the restriction site in newly synthesized strand is resistant to cleavage.

15. Loop Mediated Isothermal Amplification (LAMP) LAMP assay – simple, rapid, specific and cost effective nucleic acid amplification developed by Eiken chemical co.ltd 4 different primers designed to recognise 6 distinct regions on the target gene and the reaction process proceeds at a constant temperature using strand displacement reaction.

16. High amplification efficiency with 10 10 times in 15 to 60 minutes No need for denaturation High specificity Cost effective

17. Primers in LAMP Primers directed against 3` side – F3c, F2c, F1c Primers directed against 5` side – B1, B2, B3 Four primers: Forward inner primer F3 primer Backward inner primer B3 primer

18. SIGNAL AMPLIFICATION Amplify the signal generated by the labelled probes bDNA – Branched DNA probes Hybrid capture – Anti DNA-RNA hybrid antibody

19. Signal amplification – used to increase the sensitivity of the probe based assays. 10 3 - 10 5 nucleic acid targets can be detected Branched DNA probe system: Target sequence is captured using a capture step  hybridization with an unlabeled probe that has two hybridisation sequences  one directed against target sequence  another hybridises with bDNA amplification number.

20. Multimer system  chemically synthesized oligonucleotide chain with a comb like backbone that can bind to several reporter probes Highly sensitive because the target nucleic acid has to bind both to the capture as well as target probes before the signals are amplified

21. PROBE AMPLIFICATION Ligase Chain reaction Q Beta Replicase

22. Ligase Chain Reaction Based on sequential rounds of template dependent ligation of two juxtaposed oligonucleotide probes Exponential amplification is achieved when two pairs of oligonucleotide probes, one complementary to the lower stand of target and other complementary to the upper strand of target are used

23. Allows the discrimination of DNA sequences differing in only a single base pair The original method employed two sets of complementary primers and repeated cycles of denaturation at 100degC and ligation at 30degC using the mesophilic T4 DNA ligase . Use of mesophilic T4 or Escherichia coli ligase has the drawback of requiring the addition of fresh ligase after each denaturation step, as well as appearance of target independent ligation products

24. PRINICIPLE: Based on the ligation of two adjacent synthetic oligonucleotide primers, which uniquely hybridise to one strand of the target DNA Applications: HPV, HSV, HIV, Myco.tb , Chlamydia, Neisseria , Listeria , Borrelia

25. Detection of pathogens by LCR Eg : in case of Listeria monocytogens , the nucleotide 1258 is A-T base pair, while in case of Listeria innocua it is G-C base pair With this single nucleotide bp changes, LCR detects the pathogenic species

26. Q beta Replicase Q beta replicase is a RNA dependent RNA polymerase derived from the bacteriophage Q-beta. The enzyme complex has four subunits  one derived from Q- beta bacteriophage and remaining three from E.coli host

27. Q beta replicase - features Effects 10,000 fold amplification of the 4200-nucleotide single stranded RNA of Q beta during a very short interval Replicates the viral genomic RNA in the presence of a vast excess of host RNA Copies entire template RNA from 3` to 5` terminus without utilising endogenous primers

28. MDV RNA Midivariant (MDV) RNA is the most extensively studied non viral substrate for Q-beta replicase into which the probe sequences are inserted

29. Advantages: Duration  2 to 3 hours Isothermal Very sensitive Simultaneous detection of multiple targets

30. Plasmid Profiling Plasmids are the extra chromosomal circular double stranded DNA found in most bacteria Each bacterium has one or several plasmids Cells are lysed and the nucleic acids are subjected to electrophoresis The size and number of plasmids can be estimated Drawback: some species may contain variable number of plasmids or even unrelated bacteria may have similar number of plasmids

31. Nucleotide sequencing For determination of the nucleotide sequence in the given DNA molecule Methods: Chemical cleavage method Chain termination method Both these are automated methods Not much role in diagnostic microbiology For structure of gene, mutations and to design primers

32. Restriction Fragment Length Polymorphism (RFLP) Polymorphism in nucleotide sequence is present in all organism Restriction sites are the strands of DNA that are specifically recognised and cleaved by restriction endonucleases Useful as a Epidemiological typing tool Ribotyping - phylogenetic classification

33. S ummary Plasmid profiling Nucleotide sequening RFLP Nucleic acid hybridisation Amplification techniques

34. References Textbook of Diagnostic Microbiology – Connie R Mahon – 3 rd edition Bailey and Scotts`s Diagnostic Microbiology - 13th edition Practical microbiology – Mackie and Mccartney – 14 th edition Molecular techiniques in clinical microbiology – www.microrao.com