Kary Banks Mullis was the American biochemist who invented the technique of Polymerase Chain Reaction (PCR) in 1984; it was a great step towards revolutionizing the science. After this, Real-Time PCR abbreviated as RT-PCR has become a useful and common technique for detection and quantification of expression of elected genes . This tool to identify PCR products in real time during the chain reaction has been remained available for the last nine years but there was a great increase during last 2 years. There has been great work in the field of genomics, medicine, cell biochemistry, biophysiology and cytogenetics underlying the phenotypic and genotypic expression of cell regulation . The recent advancements in biological sciences during the past decades assist us in deep understanding of knowledge about network of various genetic systems that perform integrated intracellular functions in separated manner, i.e., the gene regulation of phenotypic expression of under observation genotype. But here the one major point is notable that major portion of the genome is going to be unknown to researchers and interrelationships between enzymes, signaling moving substances and many small molecules is still little bit known. To have complete information about metabolic pathways, gene regulation, gene expression, transcription factors, medicine and many other small molecules is still needed .
Gene expression and regulation have been utilized mainly to understand the interrelationships between ecological pressurized or weakened phenotypes and the cellular expression systems. The specific primers detection based identification system is useful to inform us about more knowledge of the aetiology, ecology and epidemiology of living organism’s pathogenic microorganisms like in plants and animals .
In simply, we can say that laboratory techniques along with nested PCR for identification on presence or absence of microbial DNA in the extracts from many plant matrices. In order to provide different products of PCR to consumers, there are also many other methods of PCR technology . Many are dependent on the utilization of internal transcription spacer regions within the nuclear ribosomal genes clusters because they are specific loci for the designed framework of PCR-based detection tools. So, these specific clusters can be easily available by utilizing universal primers and primarily are present in higher copy number of the cell but technically many inter-specific sequences modification for the framework of species specific modified primers . This RT-PCR gives us quantitative genotyping management and detection system of single nucleotide polymorphism and allelic discrimination when a small portion of the measured sample containing the mutations. This RT-PCR is best suited for many genes observation dependent on the flourochomes and analyzing of melted curves of the amplified products. As, RT-PCR is a best tool for absolute comparisons of the all transcripts involving in the under observation tissues but here is a great problem with PCR products parameters counting . Due to less efficient RNAs extraction technologies, there is little bit less application of RT-PCR in the field of diagnostics of plants and animals cells but PCR inhibiting factors like plant polysaccharides and polyphenolics . The major error in the PCR technology programming comes due to improper ways of handling PCR. The extraction, purification and mixing of nucleic acids into different compounds used in PCR technology is a really serious step. Recently, methods used for gene expression investigations start with a template synthesis step in which nucleic acids are totally freed of sticky proteins and then they are purified. The step by step processes of PCR are very tough because every time we have to shift materials from tube to tube. So, there is loss of material during shifting which creates problems [9-12]. The extraction of DNA and RNA from any living source is very time consuming and very complex. The isolation of RNA is especially very problematic because the RNA molecule is highly sensitive to enhanced temperatures and is degenerated by RNAses, which have to be promptly inactivated leading cell lysis. There are many commercial available molecular kits for PCR. The RT-PCR machines give precise calibration of kinetics of the PCR taking place inside the tube and promptly, show the visual representation of the PCR products following each series . The data taken through RT-PCR is very accurate as compared to general PCR machine. There are commercially many companies which have the websites with complete protocols and output information investigated [14-16].
In this review paper, we discuss the common criteria and technical components of PCR technology, for fast functional genomics. Examples are also provided to show the efficacy of results of plant pathology research work and verification of targets for mammalian investigations.
Applications in Different Fields
The newly developed techniques of nucleic acid amplifications have developed the medical diagnostics. Recent technologies that permit the diagnostics of different diseases through nucleic acid amplification are becoming very standardized . The applications of these new molecular techniques in the field of diagnostics of diseases are becoming much expanded. In the medicinal field first of all, we look at the disease of Cancer . Cancer comes from the addition of genetic polymorphism and mutation and/or sporadic somatic polymorphism in DNA repair, cell cycle and growth signaling genes . We have now modern techniques of diagnostics of cancer but with surgical and medical treatment of cancer, still it remains a great cause of mortality during every year. At the earlier stage of cancer, the detection of cancer and its symptoms is very difficult to observe due to its complex multidimensional nature and heterogeneity [20-22]. However, with the invention of RT-PCR the earlier diagnostics of cancer is possible with small quantity of nucleic acids of different samples. Many of the cancerous parts of the body are detected in detection system by using different probes or by using marker genes expression. The accuracy of single marker gene is not great enough for clinical implications . By adopting a genetic expression panel, the detection of Breast Cancer, Carcinoma of bladder, colorectal cancer and other types of cancers is feasible upto a good limit which was impossible during the past few decades. Now, six out of ten applications are made for the blood cancer detection. So, we can say that RT-PCR has revolutionized the medical world .
Field of Bacteriology
Earlier antibiotic therapy was dependent on the identification of Gram stain classification method. The identification of the presence of bacterial pathogens was earlier observed by the conventional PCR but later it was boosted up by using the RT-PCR. Fluorescence hybridization probes permitted a fast observation of low amounts of bacterial DNA and a right positive and negative gram stain classification technique . RT-PCR made easier to detect the bacteria by hybridization technique without any hindrances of culturing bacteria into culture media which was a tough job before the invention of PCR. A fast detection of the pathogen will make easier to detect the pathogen and to choose the right antibiotic at the right stage which will kill the bacteria. The detection and observation of the mycobacterial infections on the earlier stages is very difficult because of lower specificity and sensitivity but on the later stages it may become sensitive and specific to be detected.
Field of Virology
Many of the protocols of RT-PCR have been made to detect and to quantify the viruses from viral infected human samples . Various techniques are developed to detect the viruses especially relevant to human diseases. The identification of HSV1 and HSV2 was made possible by using TaqMan probes and it was, we can say, alternative to the conventional PCR. Recently, an observation, differentiation and quantification between HSV1 and HSV2 genotypes were attained by using different primers and probes which are called Light cycler induced targeting HSV DNA polymerase genes . A sexually transmitted disease which is genital herpes is transmitted through virus but it is accurately detected by the RT-PCR and is documented well. Moreover, the RT-PCR has also aided to investigate the interactions between virus and host; this all is studied in the RT-PCR detection system with great efficiency . RT-PCR has a great role in detecting the chronic conditions. Normally, the patients with less immunity tend to be more infected by viruses. RT-PCR multifactorial complex tools have been developed for virus specimens’ identification .
Detection of Protozoa
As RT-PCR invention has revolutionized the molecular biology, so it has also good access to detect the parasitic Protozoa of medical field interest. RT-PCR has different protocols to detect the infections of Protozoa in humans but a disease caused by Protozoa, called Malaria remains still limited in identification by PCR due to its expensive molecular protocols . RT-PCR have newly developed techniques for identification of chagas disease, amoebic dysentery, visceral leishmaniasis, giardiasis causing long lasting gastroenteritis and toxoplasmosis in the amniotic fluid of pregnant women and in the immune compromised patients. Protozoa cause the epidemic diseases around the world which are very problematic .
Detection of Fungi
Many diseases in humans are caused by the Aspergillus species . There are many conventional methods which were used earlier to detect the fungal infections; these include cell culturing, histopathology, biochemical microscopy, PCR, probe detection, CFU quantification and identification, broth dilution and staining following by microscopic investigations . The efficiency of these methods remain slower at some moments due to less efficient tool used. Quantitative and qualitative RT-PCR tools have also been made for other fungi like Coccidioides sp., and many others .
Mycotoxins have a great role as food contaminants and they are now become a big source of bad effects on food items. To cope with such a situation, a fast and less expensive diagnostic system of food borne pathogen around the world for food pathogen detection have been developed for industry and human health . Well-organized PCR-based methods for the detection and identification of the food-borne pathogens have been developed with analytical and diagnostic efficacy. In Food science, RT-PCR has strong role to detect the pathogens as Salmonella sp, which is one the most alarming pathogen of the food products .
Food borne viral contaminations are major worldwide leading disorders in humans. There is now innovation by the developing of RT-PCR by which we can successfully investigate the presence of viruses from blood and serum . This methodology is very useful for Hepatitis B virus detection [37-40]. Other food item borne viruses are rotaviruses and gastrointestinal viruses. In shortly, we can say that the detection of these viruses directly from these food items is very difficult task
There are now advanced technologies developed for the detection of DNAs in Forensic Science. Through short tandem repeats, we can detect the DNAs of different organisms. One of the most common technologies used is PCR that permits efficient genotype knowledge and information from the specimens . Forensic science mainly depends upon the slot blot technique which is much time consuming and laborious. RT-PCR has got a high standard among the tools used in Forensic science . The DNA samples used in the investigations are checked by the fluorogenic probes and same situation was implemented on the discriminate sex. In this method, different samples of DNA are taken and they are passed through the PCR and are labeled with respective probe and then probe identifies each band of DNA [43-44]. Then, there is a computational analysis of DNA by using tannic acid which compares the amplifications efficacies of unknown DNA specimens with neat and clean standards. Moreover, DNA degradation is also investigated through forensic sciences
Justification of Microarray Consequences
RT-PCR has specific role to study the gene expression designs. This study relates to the activation of genes, signal transfer, biosynthesis machinery and metabolic pathways. Gene expression investigations made easier identification of directions of stress signaling conditions of the abiotic and biotic environments. Such conditions of house-keeping genes have been studied in Potato. The efl alpha was the most stable gene among seven tested genes in biotic and abiotic conditions. Normally, the microarray confirmation through RT-PCR is highly well comparable as compared to other methods which are usually a best assay. So, here the main point is notable that justification of microarray results through RT-PCR is better than other methods .
There is a vital association between plant and the associated microbe that forms a special relationship where the microbe is a dependent element. Early diagnosis of the attack of parasite can maintain plant health but only through RT-PCR . As the severe attack of parasite can harm the plant population and can cause epidemic situations to the population of plants. In PCR, the methodology is that we take different samples of infected plant parts and then they are mixed with the PCR elements . These parasites are further identified by PCR products computation by using different probes. Several reports are reported about the diagnosis of diseases related to plant-microbe parasitic relation. In past, there came many epidemics due to parasitic diseases like Rust and Smut etc .
In plants, there are several species within a genus which correlates with each other and there is just a small difference between these species. The difference is just at a specific site when we take the cytogenetic data of these species; there comes just a small difference among chromosomes of the different species [49-52]. So, the expression analyses of different plants are checked through gene expression in which sample is taken from the plant and is utilized in PCR. So, the PCR identifies the different products through probe hybridization and all computational arrangements are made by checking the gene expression of these products. The southern blot, northern blot and western blot techniques are used to detect different species. In all these techniques, the major data comes from the RT-PCR which detects and specifies the different species of plant .
RT-PCR has become best tool for the detection and quantification of expressing profiles of under investigation genes. This review itself shows that RT-PCR technique is a best technique to express the gene expression. This tool allows the quantitative and qualitative genotyping and shows detection of the single nucleotide polymorphism as well as Genetic variation. The RT-PCR has a great role in Medical field, Forensic science, Botany, Zoology, Mycology, Bacteriology, Virology and many other fields. In short, RT-PCR is a best tool in Molecular Biology .