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Clinical Relevance of Molecular Microbiology Journal of Human Virology & Retrovirology Clinical Relevance of Molecular Microbiology Abstract Review Article Molecular methods for the detection and characterization of microorganisms Volume 3 Issue 5 - 2016 have transformed diagnostic microbiology. Detection of microorganisms was previously laborious and time taking by conventional microbiological methods but now more rapid detection by molecular methods is possible for pathogens of Department of Microbiology, Gian Sagar Medical College & public health importance. Detection of antimicrobial resistance genes and their Hospital, India characterization by genotyping is also feasible. Detection of viral resistance gene and testing of viral load for the monitoring of antiviral therapies are possible *Corresponding author: Satish Gupte, Department of because of molecular technique and automation of molecular microbiology. This Microbiology, Gian Sagar Medical College & Hospital, review will focus on basic molecular techniques and the clinical utility of these Rajpura, India, Email: molecular methods in the management of infectious diseases. Received: August 09, 2016 | Published: August 25, 2016 Keywords: Amplification techniques; Automation; Infectious diseases; Molecular methods Abbreviations: radioactive or non-radioactive labels. The common radioactive TMA: Transcription Mediated Amplification; hybridize with the target nucleic acid. Labeling can be done using NASBA: Nucleic Acid Sequence Based Amplification; SDA: Strand radioactive labels include biotin, digoxygenin and acridinium Displacement Amplification; LAMP: Loop Mediated Isothermal esterisotopes and used addition for labeling of substrate include results 32P, in 35S, production and 125I. of Thecoloured non- transcriptaseAmplification; polymerase PCR: Polymerase chain reaction Chain Reaction; LCR: Ligase Chain Reaction; CPT: Cycling Probe Technology; RT-PCR: reverse Introduction ofproduct, target nucleicsignaling acid the can positive be microorganism hybridization from reaction the clinical [2]. specimenPreparation or of from single the stranded culture. targetThe nucleic nucleic acidacid, is the extracted source Molecular microbiology is the fastest growing discipline which chemically or enzymatically. The nucleic acid is treated to stabilize as well as preserve structural integrity and then denatured to microorganism. Rapid detection of microorganism by using derive single strands. Mixture and hybridization of target and theseplays significantmolecular methodsrole for thehas detectionrevolutionized and characterizationroutine diagnostic of probe nucleic acid - The annealing of the target nucleic acid with microbiology. Microorganisms which are fastidious, slow growing, non-viable, and non-cultivable may not be detected conditions of temperature and salt concentration is called hybridization.the corresponding Detection specific of hybridization nucleotide probe reaction under - The optimum various using molecular technique. The introduction of these molecular methods of detection of hybridization reaction are radiometric techniquesby conventional and their culture automation technique was but introduced can be inidentified the form byof i.e. by autoradiography (radioactive isotope labeled probes), PCR technology. And these automation when applied to various colorimetric (biotin or digoxygenin labeled probes), fluorometric ofstages molecular of DNA detection or RNA in extraction,the clinical microbiologyamplification laboratoryand product by (fluorescein tagged avidin or antibody) or chemiluminescence detection together with real-time PCR further increase the utility on the basis of its application into two types; (acridinium ester labeled probe) [3]. Hybridization is classified provide an overview of the some basic molecular technique and Solution phase hybridization: The aqueous environment speeds clinicalmaking applicationsit more efficient of molecular and cost-effective methods for [1]. infectious This paper diseases. will up the rate of hybridization. The target DNA is denatured and the single stranded target nucleic acid is mixed with single stranded Categorizing Molecular Techniques probes is added, unhybridized single stranded nucleic acids are Hybridization methods removed using S1 nuclease digestion and the hybridized dsDNA is recovered using trichloroacetic acid precipitation. Another common method called hybridization protection assay uses acridinium ester labeled probe to hybridize with the target. Upon theUsed ability for of identificationtwo nucleic acid of nucleicstrand eachacid derivedstrand, fromless sensitivedifferent addition of H2O2 hydroxide, the duplex emits light. This assay can than amplification methods. Hybridization methods are based on be performed in few hours, because it does not require removal of bond with each other and form a double-stranded molecule (hybrid).source that So, haveone nucleic complementary acid strand base is from sequence the known to specifically organism while the other is derived from the organism to be detected and excessSolid phase of unbound hybridization single stranded: The hybridization DNA [4]. reaction occurs on percent similarity. a solid support such as nitrocellulose or nylon membrane. a. Dot hybridization: The target (nucleic acid) from sample nucleicSteps acids involved with in known hybridization nucleotide reactions sequences are -designed Production to to release target DNA and denature it to a single strand. and labeling of single stranded probes - Probes are labeled short is affixed to a membrane in the form of dot, then processed Submit Manuscript | http://medcraveonline.com J Hum Virol Retrovirol 2016, 3(5): 00107 Copyright: Clinical Relevance of Molecular Microbiology ©2016 Ravikant et al. 2/5 The membrane is immersed into a solution containing labeled probes and allowed to hybridize. Unbound probes are washed away and the hybridized duplexes are detected typically15 seconds 10-15 to 2 minutes). minutes) TheAnnealing same cycle of primers is repeated to DNA for (55° 30-35 C, according to the nature of the reporter molecules. 1-2 mins) Extension by thermos table DNA polymerase (72° C are performed on the reaction mixture consisting of target DNA, : It utilizes two probes; an unlabeled b. Sandwich hybridization primertimes to pairs, produce thermostable approx. 109 DNA copies polymerase, / 2-3 hrs. All deoxynucleotides the steps of PCR probe that is attached to a solid support such as microtitre plate and serves to capture the target from the sample to be test tube. Thermocyclers are used for the process. There are tested. The presence of this duplex is then detected using a (dATP, dTTP, dGTP and dCTP), buffer and Mg salt in the same the target sequence. various methods to validate the PCR generated amplicons, as they labeled second probe that is specific for another portion of c. Southern blot hybridization: DNA is extracted from the serve to differentiate the target amplification from non-specific amplificationi. [4]. These methods include: most cases to demonstrate similarity between the expected of various sizes using restriction enzymes. The resultant andElectrophoresis observed size of of the amplicons. amplicons should be sufficient in fragmentsorganisms areand separatedpurified; iton is gelcut byinto electrophoresis. several fragments The fragments move along the gel according to their molecular ii. Hybridization with probes for the known sequence within weight, where the smaller fragments migrate faster and farthest through the gel than the larger fragments. The target iii. Nucleotide sequencing of amplicons. DNA is transferred on to nylon or nitrocellulose membrane. the amplified target sequence Types of PCR labeled probe. Deionized formamide and detergents such as The membrane is dipped in a hybridization fluid containing I. RT-PCR The hybridized duplexes are detected by radiometric, nucleic acid (of RNA viruses) in clinical specimen as well as SDS are used to reduce non-specific binding of the probe. to prepare (Reverse cDNA library transcriptase- of mRNA. PCR): Using Used the enzyme to detect reverse viral transcriptase, a complimentary copy of the RNA is made. A enzymatic or fluorometric methods depending on the nature primer is annealed to the template RNA and with the help of d. ofNorthern reporter blotmolecule hybridization used [3]. : the target nucleic acid is reverse transcriptase, cDNA strand is synthesised by adding single stranded RNA. In in-situ hybridization the microscopy complementary base pairs. The template RNA is removed glass slide with tissue specimen acts as the solid phase. From using the enzyme RNAse H leaving behind the newly tissue sample, nucleic acid of the pathogen to be released and denatured to a single strand with the base sequence intact. generated single stranded cDNA, which can be amplified in II. PCR.Nested PCR simultaneouslyRadio-labeled probes viewing and the fluorescent histopathological probes are structure commonly of : Used for sensitivity and specificity [6]. Nested theused specimen. [4]. One can perform molecular hybridization while primers is used to amplify a target sequence and the second PCR uses two sets of amplification primers. The first set of Clinical applications of hybridization techniques: Direct target sequence. set of primers is used to amplify a region within the first genital tract infections (N. gonorrhoeae, C. Trachomatis) by Gen III. Multiplex PCR detection of pathogens e.g. in Pharyngitis (gp-A streptococci), : In multiplex PCR, two or more unique Probe Speciation
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