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Isolation of a novel DNA aptamer against LipL32 as a potential diagnostic agent for the detection of pathogenic Leptospira

Yeoh TS, Tang TH, Citartan M

Biotechnol J, 2023 Mar;18(3):e2200418.
PMID: 36426669 DOI: 10.1002/biot.202200418

Leptospirosis is a potentially life-threatening zoonosis caused by pathogenic Leptospira and for rapid diagnostics, direct detection is desirable. LipL32 protein is the most suitable biomarker for direct detection. DNA aptamers are sought to be generated against LipL32 by Systemic Evolution of Ligands via Exponential Enrichment (SELEX). LepDapt-5a is the most potent aptamer candidate among all the candidates, as determined by direct Enzyme-linked Aptasorbent Assay (ELASA). LepDapt-5a was predicted to form a G-quadruplex structure as predicted by QGRS Mapper and validated experimentally by direct ELASA. The diagnostic potential of the aptamer was further tested on a direct and sandwich ELASA platform. A LOD of 106 mL-1 and 105 mL-1 were estimated by direct and sandwich ELASA platforms, respectively, which are within the range associated with leptospiremia levels. The dot blot assay developed was able to attain a LOD of 104 CFU mL-1 against pathogenic Leptospira, which is also within the leptospiremia level. This is the first-ever DNA aptamer and hybrid-heterodimeric aptamer constructed against LipL32. The diagnostic potentiality of the LepDapt-5a DNA aptamer was proven on three major diagnostic platforms, which are direct ELASA, sandwich ELASA, and aptamer-based dot assay.
Development of an optimization pipeline of asymmetric PCR towards the generation of DNA aptamers: a guide for beginners

Yeoh TS, Anna A, Tang TH, Citartan M

World J Microbiol Biotechnol, 2022 Jan 06;38(2):31.
PMID: 34989899 DOI: 10.1007/s11274-021-03209-w

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 biotin-streptavidin-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 different randomized ssDNA library before embarking on any SELEX studies.
Aptamers isolated against mosquito-borne pathogens

Navien TN, Yeoh TS, Anna A, Tang TH, Citartan M

World J Microbiol Biotechnol, 2021 Jul 09;37(8):131.
PMID: 34240263 DOI: 10.1007/s11274-021-03097-0

Mosquito-borne diseases are a major threat to public health. The shortcomings of diagnostic tools, especially those that are antibody-based, have been blamed in part for the rising annual morbidity and mortality caused by these diseases. Antibodies harbor a number of disadvantages that can be clearly addressed by aptamers as the more promising molecular recognition elements. Aptamers are defined as single-stranded DNA or RNA oligonucleotides generated by SELEX that exhibit high binding affinity and specificity against a wide variety of target molecules based on their unique structural conformations. A number of aptamers were developed against mosquito-borne pathogens such as Dengue virus, Zika virus, Chikungunya virus, Plasmodium parasite, Francisella tularensis, Japanese encephalitis virus, Venezuelan equine encephalitis virus, Rift Valley fever virus and Yellow fever virus. Intrigued by these achievements, we carry out a comprehensive overview of the aptamers developed against these mosquito-borne infectious agents. Characteristics of the aptamers and their roles in diagnostic, therapeutic as well as other applications are emphasized.
Fulltext Aptamers as the Agent in Decontamination Assays (Apta-Decontamination Assays): From the Environment to the Potential Application In Vivo

Bilibana MP, Citartan M, Yeoh TS, Rozhdestvensky TS, Tang TH

J Nucleic Acids, 2017;2017:3712070.
PMID: 29225967 DOI: 10.1155/2017/3712070

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

Andrew A, Navien TN, Yeoh TS, Citartan M, Mangantig E, Sum MSH, et al.

PLoS Negl Trop Dis, 2022 Feb;16(2):e0010152.
PMID: 35120141 DOI: 10.1371/journal.pntd.0010152

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.