Molecular diagnosis

Nucleic acid based methods for identification of infectious agentMolecular assays are useful for the following reasons

To identify micro organisms that are difficult to growIdentify slow growing pathogensSensitive , specific and safe

Molecular methods

HybridisationAmplification Sequencing and enzymatic digestion of nucleic acid

Nucleic acid hybridizationBased on the ability of two nucleic acid strands that have complementary base sequencs to specifically bond with each other and form a double strande molecule ( a duplex or hybrid)

One nucleic strand (the probe originated from a known organism) and the target originate from an unknown

Used in the diagnosis of gonorrhea, chlamydial infectionsHistoplasmosis, coccididoisis, Blastomycosis

Identification of bacteria using the 16S ribosomal RNA. E.g.; whipples disease (Tropheryma whipplei)

Amplification (polymerase chain reaction) PCRAmplifies single copies of DNA a million foldSample incubated with primer (short pieces of DNA single strand complementary to the gene sequence to be amplified)NucleotidesTaq polymerase enzyme

DNA strands are denatured at high temperatures, and the primers allowed to hybridize, the Taq polymerase then adds nucleotides and copies the strand. The newly synthesized strand is again denatured and the process repeated many 20 to 40 times to amplify the sequence a millionfold

Steps Extraction of target nucleic acidAmplification of targetDetection of the target

Specimen with DNAExtract pure DNA using chemicals, reagents

Amplify the DNA in a thermocycler

Detect amplified by gel electrophoresis and transillumination

applicationsDiagnosis of Enteroviral meningitisHSV encephalitisHIV diagnosis and managementHCV diagnosis and managementCMV transplant and HIV patientsEBV and post transplant lymphoproliferative disorderRespiratory virusesPML associated with JC virus and AIDSBK virus in transplant patientsGonorrhea, chlamydial infections Extrapulmonary tuberculosisBordetella pertussis infection

RT-PCRReverse transcriptase PCRA variation of PCRUse of reverse transcriptase of retroviruses to synthesis cDNA from viral RNA or mRNA

REALTIME PCRUsed for quantitation of DNA and RNA after it is converted to DNA by reverse transcriptaseUseful for quantization the number of HIV genomes in a patients blood to evaluate the course of the disease and antiviral drug efficacy

Fluorescent labelled probes

Restriction enzyme analysisUsed in typing bacteriaTracing hospital outbreaksDrug resistant bacteria

B & C similar source?

Recombinant DNA techniques

Microarrayin studying gene expression

*****************