Molecular Diagnosis

Dr.M.MalathiPostgraduate II year

Department of MicrobiologyChengalpattu medical college

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.

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

How it works??

• Every organism contains some unique, species specific DNA sequences

• Molecular diagnostics makes the species specific DNA visible

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

• 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

Techniques used

• Nucleic acid hybridisation• Amplification techniques• Plasmid profiling• Nucleotide sequencing• Restriction Fragment Length Polymorphism• Pulse Field Gel electrophoresis

TARGET AMPLIFICATION

• PCR Based

• Non PCR based (Isothermal) NASBA

TBA SDA LAMP

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

• 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

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

Transcription Based 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

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

• 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.

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.

• High amplification efficiency with 1010 times in 15 to 60 minutes

• No need for denaturation• High specificity• Cost effective

Primers in LAMP

• Primers directed against 3` side – F3c, F2c, F1c• Primers directed against 5` side – B1, B2, B3

• Four primers:1. Forward inner primer2. F3 primer3. Backward inner primer4. B3 primer

SIGNAL AMPLIFICATION

• Amplify the signal generated by the labelled probes

• bDNA – Branched DNA probes

• Hybrid capture – Anti DNA-RNA hybrid antibody

• Signal amplification – used to increase the sensitivity of the probe based assays.

• 103 - 105 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.

• 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

PROBE AMPLIFICATION

• Ligase Chain reaction

• Q Beta Replicase

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

• 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

• 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

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

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

Q beta replicase - features

1. Effects 10,000 fold amplification of the 4200-nucleotide single stranded RNA of Q beta during a very short interval

2. Replicates the viral genomic RNA in the presence of a vast excess of host RNA

3. Copies entire template RNA from 3` to 5` terminus without utilising endogenous primers

MDV RNA

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

Advantages:• Duration 2 to 3 hours• Isothermal• Very sensitive• Simultaneous detection of multiple targets

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

Nucleotide sequencing

• For determination of the nucleotide sequence in the given DNA molecule

• Methods:1. Chemical cleavage method2. Chain termination method

Both these are automated methods• Not much role in diagnostic microbiologyFor structure of gene, mutations and to design

primers

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 a1. Epidemiological typing tool2. Ribotyping - phylogenetic classification

Summary

• Plasmid profiling• Nucleotide sequening• RFLP• Nucleic acid hybridisation• Amplification techniques

References

• Textbook of Diagnostic Microbiology – Connie R Mahon – 3rd edition

• Bailey and Scotts`s Diagnostic Microbiology - 13th edition

• Practical microbiology – Mackie and Mccartney – 14th edition

• Molecular techiniques in clinical microbiology – www.microrao.com