Generation And Characterization Of Rna Aptamer Against rHuEPO-a By Selex Technology

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) enables the isolation of aptamer (ssDNA or RNA that possesses high binding affinity and specificity against virtually any target molecules). In this study, a RNA aptamer against recombinant human EPO-alpha (rHuEPO-α) was successfully isolated by SELEX technology. After 11 cycles of SELEX, cloning and sequence analysis showed an appearance of a single major clone constituting the putative RNA aptamer, termed REPORA-6, with a dissociation constant of 25±1 nM

Aptamers as the Agent in Decontamination Assays (Apta-Decontamination Assays): From the Environment to the Potential Application In Vivo

The binding specificity and affinity of aptamers have long been harnessed as the key elements in the development of aptamerbased assays, particularly aptasensing application. One promising avenue that is currently explored based on the specificity and affinity of aptamers is the application of aptamers in the decontamination assays. Aptamers have been successfully harnessed as the decontamination agents to remove contaminants from the environment and to decontaminate infectious elements. The reversible denaturation property inherent in aptamers enables the repeated usage of aptamers, which can immensely save the cost of decontamination. Analogous to the point-of-care diagnostics, there is no doubt that aptamers can also be deployed in the point-ofcare aptamer-based decontamination assay, whereby decontamination can be performed anywhere and anytime for instantaneous decision-making. It is also prophesied that aptamers can also serve more than as a decontaminant, probably as a tool to capture and kill hazardous elements, particularly pathogenic agents

Development of an optimization pipeline of asymmetric PCR towards the generation of DNA aptamers: a guide for beginners

Asymmetric PCR is one of the most utilized strategies in ssDNA generation towards DNA aptamer generation due to its low cost, robustness and the low amount of starting template. Despite its advantages, careful optimization of the asymmetric PCR is still warranted to optimize the yield of ssDNA. In this present study, we have developed an extensive optimization pipeline that involves the optimization of symmetric PCR initially followed by the optimization of asymmetric PCR. In the asymmetric PCR, optimization of primer amounts/ratios, PCR cycles, annealing temperatures, template concentrations, Mg2+/dNTP concentrations and the amounts of Taq Polymerase was carried out. To further boost the generation of ssDNA, we have also integrated an additional single-stranded DNA generation method, either via lambda exonuclease or biotinstreptavidin-based separation into the optimization pipeline to further improve the yield of ssDNA generation. We have acquired 700±11.3 and 820±19.2 nM for A-PCR-lambda exonuclease and A-PCR-biotin-streptavidin-based separation, respectively. We urge to develop a separate optimization pipeline of asymmetric PCR for each diferent randomized ssDNA library before embarking on any SELEX studies

Generation of DNA aptamers against envelope 2 (E2) protein of Chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (SELEX) for diagnostic application

Abstract Introduction: Chikungunya virus (CHIKV) causes febrile illnesses in human and these cases have rapidly expanded across the globe in recent years. The current antibody-based tests for CHIKV such as ELISA have a variety of limitations associated with the molecules such as batch-to-batch variation, high cost and less stable. Aptamers are single-stranded DNA or RNA that have high affinity and specificity against a wide variety of target molecules. Compared to antibodies, aptamers are cheaper, produced in vitro, no batch-to-batch variations and thus serve as a good molecular recognition element for the development of diagnostic tests for CHIKV. Methods: Cloning, expression and purification of the recombinant CHIKV E2 was carried out and its identity was verified with western blot analysis. The purified protein was subjected to 9 SELEX cycles, the resulting nucleic acid pools were cloned and sent for sequencing. The secondary structure of the aptamer was predicted using Mfold web server and the performance of the aptamer was determined by enzyme-linked aptamer assay (ELAA). Result: The 24kDa recombinant E2 proteins were successfully cloned and purified. The protein was reactive against anti-CHIKV positive sera and anti-CHIKV polyclonal antibody with no cross reactivity with anti-dengue positive pool sera. Sequencing result revealed there were 6 potential candidates of DNA aptamers. DNA aptamer candidate with the highest frequency (61.9%) showed two loops in their predicted secondary structures. ELAA analysis revealed a binding affinity (Kd) of 177.5 nM and limit of detection was 3.3 nM. Conclusion: DNA aptamers were successfully generated and it has great potential as a feasible tool in CHIKV detection. Keywords: Chikungunya virus, E2 protein, DNA aptamer, SELE

Aptamers as the Agent in Decontamination Assays (Apta-Decontamination Assays): From the Environment to the Potential Application In Vivo

The binding specificity and affinity of aptamers have long been harnessed as the key elements in the development of aptamer-based assays, particularly aptasensing application. One promising avenue that is currently explored based on the specificity and affinity of aptamers is the application of aptamers in the decontamination assays. Aptamers have been successfully harnessed as the decontamination agents to remove contaminants from the environment and to decontaminate infectious elements. The reversible denaturation property inherent in aptamers enables the repeated usage of aptamers, which can immensely save the cost of decontamination. Analogous to the point-of-care diagnostics, there is no doubt that aptamers can also be deployed in the point-of-care aptamer-based decontamination assay, whereby decontamination can be performed anywhere and anytime for instantaneous decision-making. It is also prophesied that aptamers can also serve more than as a decontaminant, probably as a tool to capture and kill hazardous elements, particularly pathogenic agents

Current and Potential Developments of Cortisol Aptasensing towards Point-of-Care Diagnostics (POTC)

Anxiety is a psychological problem that often emerges during the normal course of human life. The detection of anxiety often involves a physical exam and a self-reporting questionnaire. However, these approaches have limitations, as the data might lack reliability and consistency upon application to the same population over time. Furthermore, there might be varying understanding and interpretations of the particular question by the participant, which necessitating the approach of using biomarker-based measurement for stress diagnosis. The most prominent biomarker related to stress, hormone cortisol, plays a key role in the fight-or-flight situation, alters the immune response, and suppresses the digestive and the reproductive systems. We have taken the endeavour to review the available aptamer-based biosensor (aptasensor) for cortisol detection. The potential point-of-care diagnostic strategies that could be harnessed for the aptasensing of cortisol were also envisaged

RNomic identification and evaluation of npcTB_6715, a nonprotein- coding RNA gene as a potential biomarker for the detection of Mycobacterium tuberculosis

Technological advances in RNA biology greatly improved transcriptome profiling during the last two decades. Besides the discovery of many small RNAs (sRNA) that are involved in the physiological and pathophysiological regulation of various cellular circuits, it becomes evident that the corresponding RNA genes might also serve as potential biomarkers to monitor the progression of disease and treatment. sRNA gene candidate npcTB_6715 was previously identified via experimental RNomic (unpublished data), and we report its application as potential biomarker for the detection of Mycobacterium tuberculosis (MTB) in patient samples. For proof of principle, we developed a multiplex PCR assay and report its validation with 500 clinical cultures, positive for Mycobacteria. The analysis revealed 98.9% sensitivity, 96.1% specificity, positive and negative predictive values of 98.6% and 96.8%, respectively. These results underscore the diagnostic value of the sRNA gene as diagnostic marker for the specific detection of MTB in clinical samples. Its successful application and the general ease of PCR-based detection compared to standard bacterial culture techniques might be the first step towards ‘point-of-care’ diagnostics of Mycobacteria. To the best of our knowledge, this is the first time for the design of diagnostic applications based on sRNA genes, in Mycobacteria

Analytical and Clinical Evaluation of a TaqMan Real-Time PCR Assay for the Detection of Chikungunya Virus

Due to the general symptoms presented by the Chikungunya virus(CHIKV)-infected patients, a laboratory test is needed to differentiate CHIKV from other viral infections. The reverse transcription-quantitative real-time PCR (RT-qPCR) is a rapid and sensitive diagnostic tool, and several assays have been developed for detecting and quantifying CHIKV. Since real-time amplification efficiency varies within and between laboratories, an assay must be validated before being used on patient samples. In this study, the diagnostic performance of a TaqMan RT-qPCR assay was evaluated using synthetic RNA and archived patient samples. The cutoff quantification cycle (Cq) value for the assay was determined by experimental evidence. We found the in-house assay was highly sensitive, with a detection limit of 3.95 RNA copies/reaction. The analytical specificity of the assay was 100%. The analytical cutoff Cq value was 37, corresponding to the mean Cq value of the detection limit. Using archived samples characterized previously, the sensitivity and specificity of the assay were 76% and 100%, respectively. The in-house assay was also compared with a commercial assay, and we found that the in-house assay had higher sensitivity. Although further evaluation with prospective patient samples is needed in the future, this validated RT-qPCR was sensitive and specific, which shows its potential to detect CHIKV in clinical sample

Diagnostic accuracy of serological tests for the diagnosis of Chikungunya virus infection: A systematic review and meta-analysis

Background Chikungunya virus (CHIKV) causes febrile illnesses and has always been misdiagnosed as other viral infections, such as dengue and Zika; thus, a laboratory test is needed. Serological tests are commonly used to diagnose CHIKV infection, but their accuracy is questionable due to varying degrees of reported sensitivities and specificities. Herein, we conducted a systematic review and meta-analysis to evaluate the diagnostic accuracy of serological tests currently available for CHIKV. Methodology and principal findings A literature search was performed in PubMed, CINAHL Complete, and Scopus databases from the 1st December 2020 until 22nd April 2021. Studies reporting sensitivity and specificity of serological tests against CHIKV that used whole blood, serum, or plasma were included. QUADAS-2 tool was used to assess the risk of bias and applicability, while R software was used for statistical analyses. Thirty-five studies were included in this meta-analysis; 72 index test data were extracted and analysed. Rapid and ELISA-based antigen tests had a pooled sensitivity of 85.8% and 82.2%, respectively, and a pooled specificity of 96.1% and 96.0%, respectively. According to our meta-analysis, antigen detection tests serve as a good diagnostic test for acute-phase samples. The IgM detection tests had more than 90% diagnostic accuracy for ELISA-based tests, immunofluorescence assays, in-house developed tests, and samples collected after seven days of symptom onset. Conversely, low sensitivity was found for the IgM rapid test (42.3%), commercial test (78.6%), and for samples collected less than seven of symptom onset (26.2%). Although IgM antibodies start to develop on day 2 of CHIKV infection, our meta-analysis revealed that the IgM detection test is not recommended for acute-phase samples. The diagnostic performance of the IgG detection tests was more than 93% regardless of the test formats and whether the test was commercially available or developed in-house. The use of samples collected after seven days of symptom onset for the IgG detection test suggests that IgG antibodies can be detected in the convalescent-phase samples. Additionally, we evaluated commercial IgM and IgG tests for CHIKV and found that ELISA-based and IFA commercial tests manufactured by Euroimmun (Lübeck, Germany), Abcam (Cambridge, UK), and Inbios (Seattle, WA) had diagnostic accuracy of above 90%, which was similar to the manufacturers’ claim. Conclusion Based on our meta-analysis, antigen or antibody-based serological tests can be used to diagnose CHIKV reliably, depending on the time of sample collection. The antigen detection tests serve as a good diagnostic test for samples collected during the acute phase (≤7 days post symptom onset) of CHIKV infection. Likewise, IgM and IgG detection tests can be used for samples collected in the convalescent phase (>7 days post symptom onset). In correlation to the clinical presentation of the patients, the combination of the IgM and IgG tests can differentiate recent and past infections

Amino acid analysis of lipases from oil pollutant isolates: Cunninghamella verticillata and Geotrichum candidum

Lipase is an enzyme commonly used in food, dairy, and other industries. Fungal lipases are more commonly used due to their secretion and easier production. Analyses of the amino acid composition of microbial lipases will hasten their potential usage in industrial applications. In this study, the major amino acid compositions of lipases secreted by oil pollutant isolates (Cunninghamella verticillata and Geotrichum candidum) enriched with fatty substances were analyzed by high performance liquid chromatography. Among eight major amino acids found in these lipases, histidine and ornithine were predominant. Lysine was absent from lipase generated by C. verticillata, while glutamine was absent from that produced by G. candidum. Conversely, glutamic acid, asparagine, histidine and arginine were present in slightly higher amounts in G. candidum. However, a slight decrease in aspartic acid and ornithine was observed in G. candidum. Analyses of the amino acids composition of lipase can potentially facilitate to predict the nature of this enzyme