Clinical Relevance of Molecular Microbiology

Journal of Human Virology & Retrovirology

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 nucleic signaling acid the can positive be microorganism hybridization from reaction the clinical [2]. specimenPreparation or offrom single the stranded culture. targetThe nucleic nucleic acid acid, 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 methods role 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 probereaction 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 butintroduced can be in identified 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 microbiology ampliﬁcation laboratory and 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 applications it 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 each acid derived strand, from less 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 inknown 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

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 2minutes). minutes) The Annealing 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 are and separated purified; iton is gel cut by into 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 of microorganisms Identification of culture singletarget sequencesspecimen. canThe be primers amplified are simultaneously. so designed thatMultiplex each isolates e.g. Identification of dimorphic fungi, mycobacteria etc by PCR can also be used to test for different organism on a AccuProbeAmplification [5]. Techniques amplification product is a unique size allowing detection and : These systems amplify the target to large Target amplification IV. identificationCompetitive ofquantitative specific organisms. PCR numbers. Some of these systems are: of nucleic acid in the sample. It (QPCR): involves Competitive a competition PCR a. betweenis a quantitative the target PCR, nucleic which acidis used and to the quantify competitive the amount DNA methods: there is no need for thermocycler - Commonly usedPCR-thermocycler methods are- is required. Isothermal amplification the same primer pair and is added in known concentration. b. Itfor differs amplification from the process.target DNA The in competitivesize or presence DNA of requires unique restriction site which is required to accurately differentiate NASBA (Nucleic acid sequence based amplification) / TMA c. (Transcription mediated amplification) the competition for the use of the primers. So, the amount oftarget the DNAtarget from DNA competitor. can be estimated Competitive by comparing PCR occurs with due the to d. SDA (Strand displacement amplification), concentration of DNA competitor. PolymeraseLAMP (Loop Chain mediated Reaction isothermal amplification). minute quantities of DNA or RNA to V. Real time PCR : PCR can enzymatically amplify large number of copies in a short period hence provides are formed (i.e.: Theydetection have of the amplicons ability to in detect real time). amplified By target DNA by fluorescently labeled probes as the hybrids ample target that can be readily detected. The basic steps in PCR plotting the increase in fluorescence versus cycle number, are: Denaturation of DNA to single strands (94° C or higher for the system produces amplification plots that provide

Citation: Ravikant, Gupte S, kaur M (2016) Clinical Relevance of Molecular Microbiology. J Hum Virol Retrovirol 3(5): 00107. DOI:

(a) Branched DNA (bDNA) probe system: The key to assay and product detection can be accomplished in one reaction vesselquantitative without picture ever ofopening the PCR hence process. cross -Both contamination amplification is which can bind to three labeled probe i.e. capture probe, lessened. -Accumulation of amplicons is monitored as it is captureis amplifier extendor molecule (unlabelled) with identical probe and branches labeled each probe. of generated, hence able to quantitative the amount of product The required nucleic acid target sequence is captured and also determine the number of copies of target in the and hybridized with an unlabeled probe that has two hybridization sequences, one directed against the target sequence and the other capable of hybridizing with bDNA original specimen. Currently fluorescent probes are used for probesamplicons used detection. are Taqman SYBR Greenprobes, I isMolecular non-specific beacons and binds and because the target nucleic acid has to bind both to the FRETto dsDNA (Fluorescent (both specific resonant and energy non- transfer) specific). hybridization Fluorescent captureamplification as well multimer. as target This probes system before is highlythe signals sensitive are

of targets present in the original sample. VI. probesHelicase-dependent [7]. amplification: This technique is amplified. Amplified signal is directly related to the number (b) Nucleic Acid Capture Assay (NACA): Here the target rather than cycling through denaturation and annealing/ molecule is directly captured onto the solid support and no extensionsimilar to traditionalcycles. DNA PCR, Helicase, but uses an a enzyme constant that temperature unwinds branched oligodeoxyribonucleotides are used for detection DNA, is used in place of thermal denaturation. and hence found more sensitive than bDNA assays. 1. Transcription mediated amplification (TMA)/ Nucleic (c) Probe amplification technique Acid Sequence Based amplification (NASBA): These are methods amplify products containing only the probe : Probe amplification technique uses three enzymes in the reaction mixture; reverse technology etc. transcriptase,isothermal RNA RNAse amplification H and DNA methods dependent rather RNA than polymerase. DNA. The sequence. It includes Ligase Chain Reaction, Cycling Probe In TMA the enzyme used for reverse transcription also has on sequential rounds of template dependent ligation of two intrinsic RNAse H activity. So, in TMA only 2 enzymes used Ligase Chain Reaction (LCR): LCR amplification is based while in NASBA, 3 enzymes used. In this technique, the cDNA of DNA sequences differing in only a single base pair. Single is created using reverse transcriptase and this is used as a strandedjuxtaposed target oligonucleotide DNA is incubated probes. LCRwith allows oligonucleotide the discrimination probes template for transcription. A cDNA copy of the target RNA is that bind to the target in an end-to-end fashion. A thermostable Reverse transcriptase builds a complementary DNA, resulting resulting duplex is heated to separate the target DNA and the inmade the formationusing a specific of RNA-cDNA primer hybrid.that binds RNAse to theenzyme target digests RNA. ligatedDNA Ligase probes. then Both ligates the separated (or joins) target the two sequence probes and together. the ligated The and removes the RNA strand from the hybrid leaving behind probes now act as targets for the probes, which bind in an end-to- single strand of cDNA. The second primer binds to the cDNA end fashion. These steps are repeated several times resulting in strand and the reverse transcriptase enzyme extends it using cDNA as template, resulting in generation of dsDNA copy of the target RNA. Using RNA polymerase, several copies geometric probe amplification [8]. of antisense RNA are produced from this dsDNA molecule. These RNA molecules can act as template for the reverse Cycling Probe Technology (CPT): It employs a DNA-RNA- transcriptase resulting in the production of dsDNA molecules other,DNA fluorescent anneals to labelledtarget DNA. probe An atenzyme a constant cuts the temperature. RNA region The of theprobe probe; labeled the probe with fluorescenceis no longer intact one end unquenching and a Quencher the signal, the 2. Strand displacement amplification (SDA): Strand and the same cycle is repeated again [7]. and not exponential. This application has been used to detect the mecAresulting gene in ofemission MRSA. of fluorescence. Probe amplification is linear nickingdisplacement its recognition amplification site and is a anpolymerase isothermal extending DNA Sequencing and Enzymatic Digestion of Nucleic Acids theamplification nick at its reaction3’ end, displacing based on athe restriction downstream endonuclease strand. It requires restriction enzyme cleavage of the DNA sample DNA sequencing means determining the order of nucleotides in a DNA molecule for which extraction of plasmid or chromosomal DNA is done. This technique can be used to study the structure prior to amplification in order to generate an amplifiable of gene, detect mutations, and compare genetic relatedness and target fragment with defined 5’- and 3’-ends. Sample DNA is to design oligonucleotide primers. Different techniques are given denatured at 95˚C in the presence of excess of four specific below: 3. primersLAMP (Loop that define Mediated target Isothermalsequence. Amplification): This method relies on auto-cycling strand displacement DNA Restriction fragment length polymorphism (or variability): In synthesis that is performed by a DNA polymerase and a set nucleotide sequence is present in all organism including microbes. of two inner and two outer primers. In the initial steps, all four primers are used, but later during the cycling reaction sites, which arise due to mutations. Restriction enzymes (RE) cut only the inner primers are used for strand displacement DNA RFLP technique relies on the base pair changes in restriction according to molecular size by gel electrophoresis Ethidium bromideDNA at specific staining 4-6 is BPused recognition to reveal sitesthe fragments and fragments under separated UV (260 Signalsynthesis Amplification: [4]. They amplify the signal generated by the nm) light. Differences resulting from base substitutions, additions, labeled probes. deletions or sequence rearrangements within RE recognition

Citation: Ravikant, Gupte S, kaur M (2016) Clinical Relevance of Molecular Microbiology. J Hum Virol Retrovirol 3(5): 00107. DOI:

sequences can be detected with this technique e.g. it can be used 3. for determination of strain variation in M. tuberculosis. performed using the Southern blot transfer technology and radioDirect isotopic probe hybridizationlabeled probe. initial Another testing approach for HPVto using was

Pulsed Field Gel Electrophoresis (PFGE) direct probes was in combination with a second blotting technique known as a dot or slot blot. This technique does to separation of large DNA fragments; hence: Inthere PFGE, is enhanced electrical not require enzyme digestion or electrophoresis and can resolutionfields are applied of fragments alternatively that differ from by fewdifferent bases. angles which lead evaluate numerous specimens by applying the nucleic acids Other molecular methods directly to a membrane with subsequent hybridization with a probe of interest. Finally, probes have also been Line Probe Assay (LIPA): applied directly to tissue sections or cells on a slide as an bound on nitrocellulose membrane/strip and amplicons is then applied to the strip which isIn just LIPA, the series reverse of probes of southern of interest blotting. are betweenin situ hybridization episomal or (ISH)cytoplasm assay viral [7]. replication The Ventana and Inform viral probes. After hybridization streptavidin labeled with alkaline HPV assay is a chromomeric ISH test that can differentiate phosphatesBiotin labeled is PCR added product and isbinds then hybridizedto the biotinylated to the immobilized hybrids. integration. Polymerase chain reaction Assays utilizing associated with HIV Mutation associated with mycobacteria target amplification, on the other hand, are based on the Clinical applications of LIPA HCV and HBV genotyping Mutation in vitro amplification of a target nucleic acid sequence. typicallyMany PCR identify assays sequence for HPV variations detection in the have E6 or used E7 genes, type- DetectRibotyping: HPV subtypes In ribotyping, medically restriction important enzymes fungi. are used to cut the genes coding for rRNA into pieces, and then separated by gel whereasspecific orthe consensusconsensus primers.primers target The type-specific conserved regions assays domains of ribosomal RNA coding sequences are used to detect the bandelectrophoresis. patterns. Clinical Universal application: probes that Allow target subtype specific differentiation conserved automationin the L1 capsid capabilities, gene. There turnaround are many time, advantages multiplexing, to the PCR technology for such screening applications, including specimen volume. ofSpoligotyping: bacterial isolates (spacer beyond oligonucleotide the species and typing) subspecies this levelstechnique [9]. sensitivity/specificity, multiple specimen types, and small- is used as typing method for M. tuberculosis. Direct Repeats Molecular Diagnostics For HIV i.e. repeated sequences of base pairs are interspersed by non- 5. The Amplicor HIV Monitor™ Assay (Roche Diagnostics, repetitive DNA spacers and these spacers are usually unique in a 4. plasma RNA is extracted with a lyses reagent containing guanidineIndianapolis, thiocyanate IN) is a and RT-PCR-based quantitation viral-load standard assay.(QS) RNA. The genome. Hence spacers are amplified and detected. It can be done onDNA nonviable Microarray: strains, It is stored a tool samplesfor analyzing or directly gene expression. on samples A [6]. DNA microarray (gene chip, DNA chip, or gene array) is a collection of HIV-1 RNA by comparison of resulting optical densities of microscopic DNA spots attached to a solid surface, such as The QS that is added into each sample permits quantification represent one gene and these are hybridized with cDNA or cRNA following amplification and detection. A 142-bp sequence in byglass, forming plastic hydrogen or silicon bonds chip between (Probes). complementary Each spot is designed nucleotide to biotinylatedthe gag gene atof HIVthe 5and ends the and QS (primers the HIV-1 SK145 and QS and amplicons SKCC1B) base pairs under high stringency conditions. A high number of are amplified by RT-PCR in a single reaction. The primers are complementary base pairs in a nucleotide sequence mean tighter captureare detected probes, in separate respectively. wells ofThe a microwell bound, platebiotinylated (MWP) a snapshot of which genes are expessed in a given cell at a given coated with HIV-specific and QS-specific oligonucleotide timenon-covalent and monitors bonding the between whole genomethe two strands.on a single Later chip. it captures Hence microarrays use relative quantitation in which the intensity of a amplicons are quantified with an avidin–horseradish feature is compared to the intensity of the same feature under a theperoxidase known input(HRP) copy conjugate number and of athe colorimetric QS RNA. TAQMAN reaction The for HRP. The HIV-1 RNA copy number is then calculated from

Applicationsdifferent condition of Molecular[7]. Techniques in Microbiology Similarnext generation to the molecular of PCR-based beacons, techniques TaqMan assays for monitoring allow for HIV-1 will utilize real-time RT- PCR (Taqman) technologies. 1. Molecular Assays For The Detection Of C. Trachomatis And N. Gonorrhoeae: Two assay formats, nucleic acid hybridization carryoveramplification contamination. and detection The toprinciple be performed of TaqMan in real-time a single closed tube, virtually eliminating amplification product the detection of these organisms. Compared to culture, the and nucleic acid amplification, have been developed for PCR is based on the cleavage of an internal probe by the 5′–3′ demonstrated improved sensitivity for detection of C. 6. exonucleaseDetection of HCVactivity of Taq polymerase during amplification. trachomatisfirst-generation and hybridizationN. gonorrhoeae. assay Second-generation (Pace-2, Gen-Probe) assays 7. HCV RNA qualitative assays determine only the presence or with even greater sensitivity have been developed. These absence of HCV RNA. Two primary methods are used in such assays: reverse transcriptase polymerase chain reaction (Digene Hybrid Capture II CT/GC) and a number of NAATs. tests include one hybridization signal amplification assay 2. Quantitative HCV RNA the methods used for qualitative cultural agents: e.g. Human papilloma virus, Hepatitis B virus HCV(RT-PCR) RNA and determination transcription- can mediated be adapted amplification to quantitative (TMA). etc.)Detection of Human Papillomavirus (Diagnosis of non- measurement by using a known amount of a synthetic

Citation: Ravikant, Gupte S, kaur M (2016) Clinical Relevance of Molecular Microbiology. J Hum Virol Retrovirol 3(5): 00107. DOI:

to assess drug resistance, Determination of resistant genes like Mec A gene, Van genes For the purpose of molecular Forstandard hepatitis and comparingC genotype the several amounts techniques of HCV amplifiedhave been to epidemiology: e.g. identify point sources for hospital and developedthe amount to of determine standard theamplified particular using genotype a calibration and subtype curve. community-based outbreaks and to predict virulence for of HCV causing infection in an individual. As discussed

and/or core regions of the HCV genome, the most highly Referencesconfirmation of culture. earlier, such techniques typically target the 5′-untranslated 1. Speers DJ (2006) Clinical Applications of Molecular Biology for Infectious Diseases. Clin Biochem Rev 27(1): 39-51. conserved regions. Because most amplification methods for 2. inHCV determining RNA also targetthe genotype. the 5′-untranslated region, qualitative PCR methods can be used to provide amplified RNA for use Detection of Cytomegalovirus Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, et al. (2006) Real-Time PCR in Clinical Microbiology: Applications for Routine 9. 3. LaboratoryAfroza B, Shahanara Testing. ClinB (2011) Microbiol Role Revof Molecular 19(1): 165-256. Biology as Diagnostic 8. tools of Infectious disease-an update. Bangladesh Journal of Medical assay are designed to detect CMV DNA; they can be either Microbiology 5(2): 27-29. qualitativePolymerase or chain quantitative reaction assays. assays andOn the the other hybrid hand, capture the NASBA assay detects pp67 mRNA, which is transcribed late William BC, Gregory JT (2005) Molecular Diagnostics. For the Clinical in the viral replicative cycle. Because the NASBA assay is nd 4. isothermal, it can detect pp67 RNA without interference of 5. Laboratorian. (2 edn), Humana Press, USA. CMV DNA. The NASBA assay is qualitative. The in-laboratory- for Diagnosis of Clinically Relevant Bacterial Infections in Blood Wendy LJH, Judith B, Cathrien AB, Petra FG (2010) Molecular Probes

developed PCR assays utilize both standard and real-time 6. Cultures. J Clin Microbiol 40(12): 4432-4438. of differences in specimen type, nucleic acid extraction beyond cag A and vac A? Helicobacter pylori genes in gastric diseases. methods,PCR methods target and gene, their primer performance sequence, varies quantitation as a result Worldda Costa J Gastroenterol DM, Pereira 21(37): Edos S, 10563-10572. Rabenhorst SH (2015) What exists standard, and detection method. 7. 10. Fastidious, slow-growing agents: e.g. Mycobacterium MicrobiologyScholz CF, Poulsen and 16S K, KilianrRNA MSequence-Based (2012) Novel MolecularMicrobiome Method Studies. for J Identiﬁcation of Streptococcus pneumoniae Applicable to Clinical 11. tuberculosis,Highly infectious Legionella agents etc. that are dangerous to culture: e.g. Francisella, Brucella, Coccidioidis immitis etc. In situ ClinEllen Microbiol Jo Baron 50(6): (2011) 1968-1973. Conventional versus Molecular Methods for detection of infectious agents: e.g. H. pylori, Toxoplasma 8. gondii etc. Pathogen Detection and the Role of Clinical Microbiology in Infection 9. Control. Journal of Clinical Microbiology 49(9suppl): S43. 12. Antiviral/ antibacterial drug susceptibility testing: e.g. HIV Assays for Infectious Diseases. Clin Microbiol Rev 2(3): 550-576. Burd EM (2010) Validation of Laboratory-Developed Molecular

Citation: Ravikant, Gupte S, kaur M (2016) Clinical Relevance of Molecular Microbiology. J Hum Virol Retrovirol 3(5): 00107. DOI:

10.15406/jhvrv.2016.03.00107