Early diagnosis and treatment is the key to identifying causal organism of disease and treat any microbial infection. In this context, diagnostic microbiology is an appropriate field to identify diverse bacterial species (1). The traditional methods of identifying bacterial species relied on culture-based method, but it could not identify all types of species (2). This process takes a lot of time and due to its selective nature, it could not reflect the presence of mixed microbial species in infections (1). However, with the advent of new molecular technologies like PCR sequencing and hybridization techniques, sensitive detection of bacteria has now become possible (2). This molecular technique is now utilized to slow growing species like Mycobacteria or difficult to culture species like Tropherymawhippelii (1). Another useful technique, Fluorescence in situ hybridization (FISH) uses rRNA-targeted oligonucleotide probes to identify individual microbial cells in situ. It gives a better result for the detection of pathogenic organism causing infection in patients with suspected sepsis (2). While FISH gave the result within one day, traditional methods took much longer time. The FISH technique was also four times sensitive than blood culture method in detecting causal organism for sepsis. Culture methods took two weeks to provide the result which FISH could give within one day (3). Thus, molecular advancement highly facilitated rapid diagnosis to improve morbidity associated with disease, reduce the length of stay at hospitals and lower the use of broad spectrum antibiotics (4). Besides this, another discovery in ISH probe is the peptic nucleic acid (PNA). It is a DNA mimic in which sugar-phosphate backbone has been replaced by the uncharged synthetic peptide backbone. This peptide backbone is resistant to hydrolytic cleavage, and so it can be utilized in imaging probes, sequence selectivity, PCR clamping and other molecular techniques (5). This new development has the potential to improve systems like the development of gene therapeutic agents and manipulating nucleic acids (6). This report reviews literature related to the description of features of peptic nucleic acid probes and oligonucleotide probes and evaluates the sensitivity of these probes in the future study.
In microbial ecology, Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes tool is highly used (10). The use of oligonucleotide in FISH is beneficial because of optimal experimental conditions which give good fluorescence signal to microbial cells containing rRNA probes. It results in the perfect matching of oligonucleotide probes and also minimizes the risk of binding of probes to non-target microorganisms (8). The optimization of oligonucleotide probes has lead to accurate evaluation of samples for methanogenic Archaea. Helper nucleotide facilitates improved in situ hybridization of six probes to three different helices (7). Thus, rRNA-targeted oligonucleotide probe has turned into an efficient tool culture-independent identification and enumeration of a variety of bacteria present in the natural environment (9). Through mapping probe accessibility models, visualization of oligonucleotides probes has become possible. It has enabled users to virtually observe the secondary and tertiary structure of ribosomal RNA in silico (9). One study on 16S rRNA of E.coli showed that about 20% signal of the maximal fluorescence signal is received in one third of 200 probes examined (7). Besides this facts, direct screening of PNA probe is very costly as the price of a single PNA probe is higher than DNA analogs by ten times (10). Thus, specificity of PNA probe is highly critical in FISH detection (10). The PNA probes can enter algal cells quickly and render the cells green by binding with the target species. Apart from this, PNA probes have high efficiency of hybridization, excellent binding capacity and competent enough for target cell enumeration. In addition to this, it provides increased binding capacity for target nucleic acid sequences. Another advantage is that PNA and LNA oligomers can specifically bind with DNA and RNA strands. There is a minimum chance of destabilization due to mismatches compared to typical oligonucleotide duplex (11). On comparison of DNA oligonucleotide sequence and PNA sequence, it was found that fluorescein-labeled PNA probes provide intensified fluorescent responses. The modern technology uses fluorescein-labeled oligonucleotides to monitor in situ hybridization to telomeric repeat sequence (12). However, more sensitive and better quantitative results can be obtained by using fluorescein-labeled PNAs.
In the absence of electrostatic repulsion also, PNA can hybridize to DNA OR RNA because of its uncharged polyamide backbone. It occurs under low salt concentration, and short PNA oligomers of 13-18 based can be used because of its greater affinity (6). PNA binds specifically with cell penetrating peptides or lipophilic molecule to increase cellular delivery during antigen application as it cannot diffuse at a fast rate (6). Oligonucleotides are designed to recognize and hybridize to those genes where there is the chance of interference with the transcription of that gene. This process is called antigen strategy (13). In another way, nucleic acids analogs are so designed to inhibit translation by binding to the complementary sequence in mRNA. Thus it exhibits anti-sense strategy. PNA is beneficial for FISH technique as it binds to DNA and RNA at a much faster rate even at low salt concentration and during unfavorable conditions (6). PNA probes are also useful for in vivo imaging of mRNA and aids in cancer research (14). PNA also plays a role in specifically binding to globin mRNA during cDNA synthesis and inhibiting reverse transcription (6). PNA can form a stable triplex structure with the DNA and arrest transcriptional processes. For this feature, they are used in microarrays and biosensors (15). A PNA microarray in combination with PCR helps in the detection of genetically modified organisms in food (16). PNA can bind to DNA and RNA sequence following Watson-Crick hydrogen bonding rules. It also enables elongation of DNA primers by DNA polymerase (13). PNA also helps in simplifying the Southern hybridization process by reducing the time because in the presence of PNA pre-gel hybridization cumbersome processes like separation, probing and washing steps are eliminated. They also act as genome cutters in combination with methylases and other restriction endonucleases (17). Because of it unique hybridization properties, PNA is also used to purify nucleic acids. PNAs can purify nucleic acid by nickel affinity chromatography. PNA inhibits elongation of primers by DNA polymerase. When PNA invades DNA duplex under physiological conditions, it inhibits DNA replication. Inhibition of replication is also possible if DNA is single-stranded (19). Moreover, biotinylated PNA molecule can isolate transcriptionally active chromatin fragments containing multiple tandem triplets (20). Thus PNA holds great promise for gene therapeutic drugs design (13). With the advancement in diagnostic technology and therapeutic products, PNAs has a bright future in the field of biotechnology (6).
The rapid identification of five major Candida species found in positive blood culture is possible by the Yeast Traffic Light PNA FISH technique. It has excellent sensitivity and specificity with very few cross-reactions (21). Measurement of methylation level in heterogeneous tissues can be done by PNA hybridization and MALDI-TOF-MS analysis. It promotes rapid evaluation of DNA methylation markers at first analysis (22). PNA can be practically applied in HPV genotyping assays because of its sensitivity, reliability and specificity and longer shelf life (23). The PNAs are also resistant to protease and nuclease degradation because of its unnatural backbone. Because of the property of resistance to enzymatic degradation, it has extended life both in vivo and in-vitro. This property of high biostability is the reasons for increase reliability of PNAs in diagnostic applications (5). The PNA array can be strategically used for the development of advanced methods that can detect several DNA targets along the food chain in a systematic way (16). The PNA-FISH assay is also a more appropriate technique for exiting Salmonella probes compared to culture-based methods. It is more sensitive than PCR and ELISA protocols (24). The use of PNA probe in FISH technique has also helped in the detection of Cronobacter in powdered infant formula (25). Due to high specificity and accuracy by FISH method, it has assisted in the discovery of new techniques for detection of Salmonella species un food samples (26). It is also more efficient way compared to culture-based methods. On the whole, it can be concluded that flow cytometry PNA-FISH is a beneficial and standardized approach for detection and identification of pathogens from blood cultures. This method will be useful in the long run for identification of microbiological diagnostics at an accelerated rate, and it will eventually improve heath outcome of patients with sepsis (27).
The invention of PNA probes has provided the biotechnological world with new testing formats for clinical microbiology and pathology testing. It has lead to the discovery of new test applications for the detection of the variety of sensitive diagnostic techniques (31). The clinical world can benefit a lot from the development of PNA probes as it will lead to better health outcome in patients. Because of its high sequence selectivity, PNA probes open new doors for evaluating the progress of cancer and planning appropriate therapy in patients. The PNA array biosensor will be highly useful in the future for identification of single-base mutations in cancer-specific genes in patients. However, this new technology dependent on the PNA-based device is still in its developmental form, and more research is required to fully understand its function and properties in the identification of specific sequences. Researchers still need to work on the diagnostic need of higher sensitivity and analyze reduced matrix effects (28). New development in the application PNA in genetics and cytogenetic also came up last years, and it demonstrated the development of the strong yet simplified technique for detecting mutation or screening aneuploidy, genome mapping, and antigen therapy. The PNA-FISH method offered the need to design PNA probes that can act against almost any pathological and non-pathological microorganism. With its specificity property, it can be applied in different sections of clinical and pathological examination and microbiological testing (29). Short PNA sequence along with different fluorochromes now constitutes a new class of genomic biomarkers for microarray platforms. Its discovery has now created another level of challenge for extending this technology to the diagnosis of preimplantation genetics (5). PNA still cannot be used as a genetic therapeutic agent because it needs development of an efficient technique for uptake and penetration of PNA probes inside the cell (13). However, despite these challenges, it has still offered great scope for the development in the field of biotechnology, biology, and chemistry. PNA molecules have offered a useful alternative that can take the place of real-time PCR probes. It has specifically led to the design of probes that can target particular defect sequence (30). Another advantage of PNA lies in the development of in vivo fluorescence imaging. The ability to insert fluorescent probes inside living cells will enable in-depth study of mRNA transfer and live gene expression (5). In vitro studies have also demonstrated that PNA could inhibit both transcription and translation of targeted and problematic genes. Therefore the agent holds a great promise antigene and antisense therapy. However, one major problem that will still occur is that delivery of PNA through cell membrane will be a challenging task. Thus, PNA-FISH is simplistic like traditional staining techniques because of its sensitivity and specificity to target sequence in molecular technologies. Thus an approach that is useful enough to replace existing methodology of microbiological testing is needed to plan appropriate therapy for patients with infection.
Thus, the report highlighted the need for early diagnosis in treating microbial infection and discussed the different technique that is used in diagnostic testing. It specifically brought into focus the advantaged of fluorescence in situ hybridization in detecting DNA sequence compared to traditional culture-based methods. The report mainly gives detail about the new development in ISH probe which is PNA. The report gave a detailed description of the mechanism by which it is resistant to hydrolytic cleavage. Due its property of specificity and sequence selectivity, this agent has found the place in antisense drug therapy, PCR clamping, and imaging probes. Comparison of oligonucleotide probe wth other PNA probes stressed the advantage of PNA probe in the identification of target sequence and inhibiting transcription and translation in those genes. Thus PNA probes have great scope for future diagnostic testing, and it can help a lot in accelerating diagnostic patients. It will give better health outcome in the long run.
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