The evaluation results of proposed antigen rapid diagnostic tests for COVID-19: some possible factors might influence.

PURPOSE: In addition to existing gold standard qRT-PCR methods, there is a need to develop reliable rapid tests for infection control with early notification of COVID-19 cases to enable effective outbreak management. We evaluated the validity of the three Ag-RDT kits proposed by some companies in different countries by using qRT-PCR and analyzed its results. METHODS: Each of the three Ag-RDT kits (namely A, B, and C) was tested with 90 samples, consisting of samples with Ct ≤ 25, samples with Ct > 25, and negative SARS-CoV-2 PCR samples. RESULTS: This study showed that for samples with Ct > 25, all the three kits could not detect SARS-CoV-2 Ag (0% sensitivity) but showed 100% specificity. Meanwhile, for samples with Ct ≤ 25, kit C was the best (76.7% sensitivity and 100% specificity). The PPV of the three kits was 100%, but their NPV ranged 63-84.8%. Kit C showed the best accuracy (89.9%). Some factors might influence the results of evaluation, such as variation of virus proteins and transportation-storage of the kits. CONCLUSION: The overall specificity of the three kits for all samples was high; however, all of them have not met the minimum performance requirements of ≥ 80% sensitivity for samples with Ct ≤ 25. The validation test is much necessary to be carried out by the authority in national health care to ensure the feasibility of the kit for point-of-care testing (POCT) of COVID-19. Some factors that might influence should be anticipated to increase their sensitivities and specificities.

Fluorescence spectrophotometry for COVID-19 determination in clinical swab samples.

Considering the limitations of the assays currently available for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its emerging variants, a simple and rapid method using fluorescence spectrophotometry was developed to detect coronavirus disease 2019 (COVID-19). Forty clinical swab samples were collected from the nasopharyngeal and oropharyngeal cavities of COVID-19-positive and -negative. Each sample was divided into two parts. The first part of the samples was analyzed using reverse transcription-polymerase chain reaction (RT-qPCR) as the control method to identify COVID-19-positive and -negative samples. The second part of the samples was analyzed using fluorescence spectrophotometry. Fluorescence measurements were performed at excitation and emission wavelengths ranging from 200 to 800 nm. Twenty COVID-19-positive samples and twenty COVID-19-negative samples were detected based on RT-qPCR results. The fluorescence spectrum data indicated that the COVID-19-positive and -negative samples had significantly different characteristics. All positive samples could be distinguished from negative samples by fluorescence spectrophotometry. Principal component analysis showed that COVID-19-positive samples were clustered separately from COVID-19-negative samples. The specificity and accuracy of this experiment reached 100%. Limit of detection (LOD) obtained 42.20 copies/ml (Ct value of 33.65 cycles) for E gene and 63.60 copies/ml (Ct value of 31.36 cycles) for ORF1ab gene. This identification process only required 4 min. Thus, this technique offers an efficient and accurate method to identify an individual with active SARS-CoV-2 infection and can be easily adapted for the early investigation of COVID-19, in general.

THE APPLICATION OF CELL-FREE FETAL DNA (cff-DNA) AND SIBLINGS DNA METHODS IN THE PROCESS OF PATERNITY TEST THROUGH CODIS STR LOCI (CSF1PO, THO1, TPOX, AND vWA).

BACKGROUND: The non-invasive cff-DNA and siblings DNA methods are the latest breakthroughs in the forensic identification process. The use of cff-DNA and siblings DNA as non-invasive techniques in the forensic identification process has, hitherto, not been widely proven. METHODS AND MATERIALS: This was an analytic observational study. The sample of this study consisted of peripheral blood of women in the second trimester of pregnancy and their two biological children. The kinship analysis was carried out through siblings' DNA and cff-DNA from the mothers through CODIS STR loci (CSF1PO, THO1, TPOX, and vWA). RESULTS: The means of allele sharing between full siblings in loci CSF1PO, THO1, TPOX, and vWA were 0 (13.75%), 1 (44.75%), and 2 (41.50%). The allele sharing found in the study is in line with the one in previous research conducted by Wenk (1998) and the theory proposed by O'Connor (2011), indicating that one allele sharing dominates, contrasting with the finding of previous research conducted by Sosiawan (2020) revealing that 2-allele sharing was more superior. The variation is caused by the ethnicity having a different genetic contribution among the population. The variation can be attributed to historical and demographical processes leading to genetic drift. CONCLUSION: The mean of SI in 1 allele sharing in CODIS STR loci (CSF1PO, THO1, TPOX, and vWA) has the highest value of 44.5%. The use of cff-DNA of pregnant women as one of the non-invasive techniques can serve as an alternative material in a paternity test.

The use of touch DNA analysis in forensic identification focusing on Short Tandem Repeat- Combined DNA Index System loci THO1, CSF1PO and TPOX.

Forensic identification through DNA analysis is an accurate diagnostic tool. Deoxyribonucleic Acid (DNA) analysis is via DNA repetitive regions with less than 1 kb base size is called 'microsatellite' or Short Tandem Repeat (STR). At the crime scene, the perpetrator's skin may accidentally be in contact with surrounding objects, thereby transferring trace evidence to the objects. In this study DNA was obtained using "touch DNA" from two buccal smears and two smear from watches and cellphones from volunteers who had signed the consent form. Samples were isolated using DNAzol. The quantity of DNA obtained will be measured using a UV spectrophotometer. For DNA amplification using 3 STR CODIS loci namely TH01, CSF1PO, and TPOX. The last step is visualization using acrylamide gel and silver staining. Mean levels of DNA (UVVisible Spectrophotometer) were 167.89±85.71 µg/mL for the buccal swab, 59.19±5.58 µg/mL for the watch swab, and 38.09±2.12 µg/mL for the mobile swab; the purity of the buccal swab DNA was 1.79±0.71, of the watch swab 1.69±0.76, and of the mobile swab 1.53±0.56. Visualization of PCR products on Polyacrylamide Agarose Composite Gel Electrophoresis stained with Silver and amplified using the standard primers THOI, TPOX and CSF1PO for STR Combined DNA Index System (CODIS) showed a 100% detection of amplicons. Both the buccal swab, watch swab and handphone swabs had trace amount of DNA that was sufficient to be isolated and amplified by using Polymerase Chain Reaction on the STR CODIS loci THO1, CSF1PO and TPOX.