IntroductionOver the past years, clinical Microbiology has become reliant on the techniques of molecular diagnostic. Frequently in the diagnosis of infectious diseases, the amplificationof nucleic acids are undergone using molecular tests 1.
Traditionally,assays based on phenotype rather than the genotypeof pathogens were used but are nowbeing replaced by current technologies in diagnostic microbiology. Some of thetraditional assays in microbiology diagnostic areserology, antigenaemia, microscopy, and microbial culture 2. However, despite theusefulness of these assays new technologies have allowed us to betterunderstand and analyse the dynamics andcomposition of microbial communities. Forexample, In past decades, human intestinal bacteria were quantified using quantitative Polymerase Chain Reaction (PCR) amplification of specific sequences 3.Polymerase ChainReaction (PCR) is a technique that was invented in 1984 by Kary Mullis, that amplifiesa region DNA or a gene.
This technique later revolutionizedscience and paved the way for RT-PCR or real-timePCR which was later invented 1996 4which allowed the product of PCR to be detectedin real time5.The visualizationof PCR product occurs in real time whenDNA detection dyes such as syber green orfluorescent probe are combined with PCR 6. The of PCR steps can be divided into threeprocesses: denaturing, annealing and extending. During denaturing,double-stranded DNA is heated above 90 C and separated into two single strands.
Then the temperature is then loweredbetween 50-60 C to allow oligonucleotideprimers to anneal to DNA template. In thethird step, the temperature is raised between 70-78 C allowing optimal primerextension; thus, creating a new strand of DNA 6.Throughout the years conventionalPCR methods have been superseded by RT PCR due to its detection of differentpathogen strains, rapid diagnosis, detection andquantification of PCR products 1. Also, the close setting ofPCR reduces the chances of contaminationand provides sensitivity and specificityin research6. These various advantages of RT PCR haveallowed the technique to become well-established and increased drastically. However,despite several improvements in the increase of accuracy,there is a limitation to the use of RT-PCR7, 8.
One of the prominent problems thatlead to the production of real-time PCR assayswas to combat the prolific spread of nosocomial pathogens. Namely,carbapenem-resistant Enterobacteriaceae (CRE), particularly E.coli and K. pneumoniae,research on CRE, multi-drug resistantA. baumannii and P. aeruginosa, and methicillin-resistantStaphylococcus aureus (MRSA) 9.The goal of this review isto discussthe application of real-time PCR inClinical microbiology with focus on microbialgenotyping, diagnosis, recent advances, microbiologicalapplications, limitation and advantages of PCR Real-time PCR quantificationSpecific Alleles and DNA aredetected and determined by the use of quantificationusing real-time PCR.8 Inquantification, the number of nucleicacids in a range of samples is detected and measured,many of which end up impacting life science, agriculture or diagnostics.
10 During quantification method,the method of choice may vary depending onthe whether the quantification needs to be relative or absolute, the targetsequence, range of mRNA and the required accuracy11.Based on the amount of templatewithin a sample the quantification caneither relative or absolute. In arelative quantification, although the approach is simple it analyses changes ina number of target sequences of gene expression in a given sample relative within the same matrix orrelative to a reference control11, 2. This approach examines the adequacy of the relativeexpression ratio and the physiological changes within a gene expression11.
On the other hand, Absolute quantificationrequires more effort. It is either based on an externalor internal cure, where known quantity isused to quantitate the unknowns. Thisapproach is used when there is no standardized reagent or an absent ofsequential sequence2.The use of real-time PCR for the quantification of the pathogen hasbecome increasingly popular for its use in pathogen diagnostic. Now, with theavailability of the amount of sequencingdata, designing micro-organisms assays has become easier8.However, to do so, it is important that the necessary background steps and the informationare understood. To quantify a sample’s template, it is imperative that to contemplate the number of controls ant the typeof sample so that the concentration canbe acutely determined. Also, to preventcompetition within a wild-type template, it is important that the internal controls are added a suitable level.
Since the internal controls are used to determine false-negatives during the quantification.Finally, when expressing the results the to express the control to a suitablebiomarker of the organism2. Quantificationusing real-time PCR has many advantages. As mention, its speed,sensitivity and specificity make it oneof the touchstones for nucleic quantification 10. Additionally, the technology can create a single reaction from themultiplex amplification of several targets as well as a wide range of dynamic forquantification (7–8 Log10). Although the multiplexingoption relies on the inclusion ofinternal application it is essential for the diagnostic and detection ofeach quantitative assay) 8. Nevertheless, the appearanceof current and improved formulae aremaking quantification easier and more reliable2..
Microbial GenotypingConstantemphases in studies associated with diseases dealing with single nucleotidepolymorphisms (SNPs) has given rise to the genotyping platform and there is nosecret this has lead to a corresponding increase in methods used to investigatediseases with SNPs association 12The identification of subtypes and variation of different pathogens at anucleic level is called genotyping . In Clinical microbiology genotyping playsan important role in managing and the risk assessment of infectious diseases1To characterizeunknown nucleic acid nucleotide sequencing is used. To reduce the lengthyprocess real-time PCR became popular androutine in declining deletion, orinsertions 2. Detection dyes such as Sybrgreen and hybprob are the two mostcommonly used in genotyping.
Although other analyses such as light-up and double-stranded oligonucleotide and hyBeacons are not commonly used in thisfunction they could also function in thesame role2.Moreover,Numerous amount of probe-based genotypingstrategies for RT PCR has been established. In the presence of specific detectionprobes, most of these methods contaiming PCR amplified DNA with polymorphismsites can be be detected. More specifically alleles in a single tubewere recognized because of probes that were labelled with fluorophores13. At endpoint analysis,genotyped data are obtained after thecompletion of PCR. Fluorophore and targetstrand complexes formation are encouragedwhen amplicon is denatured then rapidly cooled. Moderately, the temperature isrisen allowing the fluorescence from each vessel to be continuously recorded2.Genotyping is quite useful, it canbe used in phylogenic analysis, in theprovision of extensive and accurate data once it’sdirectly sequenced and it is also suitable for identicalof antimicrobial resistance and high-risk strains.
Contrary, genotyping can be a very costly and laborious method1. Also, although genotypingusing real-time PCR can be quite useful whenit comes to microbial genotyping RT PCR is not the most effective method forthe analyzation of bacterial Strain. Since there is a limitation in genotyping of bacterial strains fact, Repetitivesequence-based PCR (rep-PCR) was developed to classify subspecies and strain of bacteria14.In epidemiology study, the ultimateresolution could be provided but if the entire genome of bacterial species is genotypedbut since only small regions o of the genome is interrogate the resolution is limited. An organism with high microbial genome makes it’sit easier for healthcare centres to reconstructthe pathway which could intern control the outbreaks.A downside to this is that for eachpathogen an entire genome sequencing would have to be given to determine if itis cost effeicient15.
Food and Safety in microbiologyOver the past 25 y, there have beenconsiderable advances in thedevelopment and use of moleculartechniques for the detection ofmicroorganisms in foodstuffs as aresult of the increasing demandfor rapid results. These are normallybased on detecting specificDNA or RNA target sequences usingamplification processes,in particular the polymerase chainreaction (PCR) (Cocolin andothers 2011). Their adoption, in manyinstances, has replaced orsupplemented traditional culturedetection methods with culturemethods still recognized as the goldstandard for most bacterialfoodborne pathogens. But in the case ofsome foodborne viruses,which are not culturable, nucleicacid-based assays remain the onlythe choicefor their detection.Annually, millions of people acrossthe world in both developed and undeveloped countries have died and arecurrently dying or being infected by diseases caused by foodborne pathogens. Traditional culture methods were being been still being used in order todetect pathogen transmitted through foods. This method of detection islaborious and time-consuming16.To overcome this dilemma, a rapid, cost-effective, and automateddiagnosis of food-borne was needed to reduce health hazard from foodcontamination.
This sparked the development first culture-independent molecular method around 18 years ago2, 5. Thisallowed scientists to rely on the molecularmethods to study the micro-organisms in their environment. Now, there has beenmore developed advances using molecular techniques to detect micro-organisms infoods17. One of the techniques mostlyused to detect, identify and quantify foodborne pathogen is real-time PCR2.Even though real-time PCR methods have replaced most traditional culture methodsthe culture method is still useful andare still used to detect most bacterial foodborne pathogens.
Although somefoodborne pathogens such as bacteria can be detected by the use of culture,other foodborne pathogens such as viruses cannot 17. Infections caused byfoodborne viruses is one of the world’s human dominating diseases. In the Hepatitis B virus alone, approximately350 are carriers and two billion peoplebeing past carriers. The development of nucleic acid assay based methods suchas real-time PCR remains quite useful in the detection of viruses such asHepatitis B and others5. This is because use ofreal-time PCR, the application process isutilized in order to detect specific RNA or DNA sequences 17Apart from Hepatitis B, there arenumerous other foodborne pathogens such as Mycotoxins are another major concernin food contamination worldwide because of a negativeeffect on human population 5.These infectious pathogensonly aided in the developmental use of PCR methods throughout the years.Allowing guidelines to be established by ISO standards are used in thequantification of foodborne pathogens2.The use of real-time PCR hasnumerous advantages in the use of foodmicrobiology.
It is accurate in the diagnosis of diseases,has a low carryover contamination, user-friendlyinterpretation, and as suggested beforeit is able to combine detection and amplification into one step5.Despite the usefulness of real-time PCR has its limitations. For example,quantification of bacteria in raw foods ofsome viruses remains a difficult task. Also,the technology has the inability differentiatebetween live and dead cells and detects asmall number of bacteria. This inabilityto pick up on these may result inpathogenic bacteria ending up in processedfoods. In order to combat this problem pre- enriched agar could be used prior to real-time PCR to aid in its detection5. This will allow multiplicationof bacteria for detection. Taxonomy andDiagnosisMicrobial taxonomy seeks to accurately identify and monitor pathogens such as viruses, bacteria or parasites18.
The importance ofidentification is essential inmicrobiology because it provides diagnosisand treatment diseases in either, human animals and plants19.In taxonomy the polyphasic approachis being utilized in order tocollectively determine the position of an isoatebase on phenotypic and genotypic properties Also in recent years, sequences like 16S rRNA gene sequencing have been used for identification of microorganism and determiningtheir evolutionary relationship20,19.However, it was not until the early 90s that the useof mathematics and computational studies determined that each genome has specific-specific signatures. These signature are now what allow science to determinethe which species are dissimilar or related based on these signatures19Approximately 70% of medicaldecision is analysed in the lab, some ofwhich are clinical microbiology techniques are utilised. For example, sepsis culture-basedtechniques remains a standard method of detection21. In theUnited States, bloodstream infectionkills around 14-50 % of patients and now it is oneof the 11 leading causes of death18. The lengthytime of culture-basedmethod is inconvenient since most of the diagnosis and treatment are not begiven in the 6-hour window; thus,mortality and morbidity increases.
However, with the use of real-timePCR results were able to be interpreted at a faster rate and reduced the amountof time and the application was also able to tell the severity of the infection, an abundanceof each pathogen, and quantify polymicrobial pathogens to initiate a treatment for the infection20, 22.However,unlike viruses that might solely on the use of real-time PCR or other PCR basedassay viruses and fungi use thecombination of culture-based methods and PCR based methods to come up with adiagnosis and the treatment of infections caused by thesepathogens with the exception of Chlamydia, methicillin-resistant Staphylococcusaureus (MRSA), and drug-resistant Mycobacterium tuberculosis 15. Other advantages andDisadvantagesAs mentioned before real-time PCR reduced cross-contamination,saves time, has a high sensitivity and specificitythroughout1, 23.Contrary to the advantage of real-time PCR there are many otherlimitations. The technology has the inability ifthe express gene is detected in a cell, tissue and the comparison among differentconditions cannot be compared24. When compared to conventional PCR, real-time PCR has it has restricted multiples capabilitiesand is very incompatible with some fluorescentchemistries platform2.
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