scholarly journals Feasibility of Rapid Diagnostic Technology for SARS-CoV-2 Virus Using a Trace Amount of Saliva

Diagnostics ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 2024
Author(s):  
Reiko Tokuyama-Toda ◽  
Masaaki Muraoka ◽  
Chika Terada-Ito ◽  
Shinji Ide ◽  
Toshikatsu Horiuchi ◽  
...  

Containment of SARS-CoV-2 has become an urgent global issue. To overcome the problems of conventional quantitative polymerase chain reaction (qPCR) tests, we verified the usefulness of a mobile qPCR device that utilizes mouthwash to obtain a saliva sample with the aim of developing a rapid diagnostic method for SARS-CoV-2. First, we examined whether anyone could easily operate this device. Then, we examined whether RNA in the mouthwash could be detected in a short time. In addition, we investigated whether it was possible to diagnose SARS-CoV-2 infection using mouthwash obtained from COVID-19 patients undergoing hospitalization. The results revealed that all subjects were able to complete the operation properly without error. In addition, RNase P was detected in the mouthwash without pretreatment. The average detection time was 18 min, which is significantly shorter than conventional qPCR devices. Furthermore, this device detected SARS-CoV-2 in the mouthwash of a COVID-19 patient undergoing hospitalization. The above findings verified the efficacy of this diagnostic method, which had a low risk of infection, was technically simple, and provided stable results. Therefore, this method is useful for the rapid detection of SARS-CoV-2.

2019 ◽  
Author(s):  
Jade Benjamin-Chung ◽  
Nils Pilotte ◽  
Ayse Ercumen ◽  
Jessica R. Grant ◽  
Jacqueline R.M.A. Maasch ◽  
...  

AbstractAn active area of research investigates whether soil-transmitted helminths (STH) can be locally eliminated in endemic settings. In such settings, highly sensitive diagnostics are needed to detect STH infection. We compared double-slide Kato-Katz, the most commonly used copromicroscopic detection method, to multi-parallel quantitative polymerase chain reaction (qPCR) in 2,800 stool samples from children 2-12 years in rural Bangladesh. We estimated the sensitivity and specificity of each diagnostic using Bayesian latent class analysis. Compared to Kato-Katz, STH prevalence using qPCR was almost 3-fold higher for hookworm species and nearly 2-fold higher forTrichuris trichiura.Ascaris lumbricoidesprevalence was lower using qPCR, and 26% of samples classified asA. lumbricoidespositive by Kato-Katz were negative by qPCR. Amplicon sequencing of the 18S rDNA from 10 samples confirmed thatA. lumbricoideswas absent in samples classified as positive by Kato-Katz and negative by qPCR. The sensitivity of Kato-Katz was 49% forA. lumbricoides, 32% for hookworm, and 52% forT. trichiura; the sensitivity of qPCR was 79% forA. lumbricoides, 93% for hookworm, and 90% forT. trichiura. Specificity was ≥ 97% for both tests for all STH except for Kato-Katz forA. lumbricoides(specificity = 68%). There were moderate negative, monotonic correlations between qPCR cycle quantification values and eggs per gram quantified by Kato-Katz. While it is widely assumed that Kato-Katz has few false positives, our results indicate otherwise. Our findings suggest that qPCR is more appropriate than Kato-Katz in low intensity infection settings because of its higher sensitivity and specificity.Author summarySoil-transmitted helminth infections (STH) (e.g.,Ascaris, hookworm,Trichuris) contribute to a large burden of disease among children in low- and middle-income countries. There is increasing interest in implementing large-scale deworming programs to eliminate STH in certain settings. Efforts to monitor whether local elimination has occurred require sensitive diagnostic tests that will not miss positive cases. Kato-Katz, a microscopy-based diagnostic test, has commonly been used to identify STH eggs in stool, but in settings where infection intensity is low, this method frequently misses positive samples because it requires visual identification of small numbers of eggs, and eggs may degrade prior to visualization. Quantitative polymerase chain reaction (qPCR) is a molecular diagnostic method that may miss fewer infections because it identifies STH DNA in stool, which can be detected in very small quantities and is less likely to degrade. This study compared the performance of Kato-Katz and qPCR using 2,800 stool samples from children aged 2-12 years in rural Bangladesh. qPCR detected substantially more hookworm andTrichurisinfections than Kato-Katz. 26% of samples were classified asAscarispositive by Kato-Katz and negative by qPCR. We conclude that qPCR is a more appropriate diagnostic method than Kato-Katz in low infection intensity settings.


2014 ◽  
Vol 8 (3) ◽  
pp. 399-403
Author(s):  
Chinnuwat Sanguansermsri ◽  
Pranoot Tanpaiboon ◽  
Pimlak Charoenkwan ◽  
Arunee Phusua

Abstract Background: Trisomy 21 or Down syndrome (DS) is the most common aneuploidy disorder. Fetal karyotypic analysis remains the criterion standard for prenatal diagnosis of DS, although the method is time consuming and requires skilled personnel. Real-time quantitative polymerase chain reaction (qPCR) can be used to determine a difference in the amount of gene copy by calculation of the difference between the cycle threshold (ΔCT) of a tested gene and a reference gene. Objectives: To develop a rapid qPCR diagnostic method for trisomy 21. Methods: Ten DS patients with the known karyotype of trisomy 21 were enrolled. Their parents were included as controls. D21S11 locus on chromosome 21 and SM locus on chromosome 16 from each subject were amplified by qPCR. The D21S11/SM ΔCT and 2-ΔΔCT values were compared between DS patients and their parents. Results: The D21S11/SM ΔCT values of the DS patients were higher than their respective controls except for one family. The mean 2-ΔΔCT value between patients and mothers was 1.88 ± 0.95 (95% CI 1.20-2.56), and between fathers and mothers as controls was 1.06 ± 0.68 (95% CI 0.58-1.54). Conclusion: The diagnostic method of trisomy 21 by using qPCR is feasible, although false negative results may occur. Using more index genes is recommended to increase the sensitivity and specificity.


Bioanalysis ◽  
2021 ◽  
Author(s):  
Kaichen Huang ◽  
Hailing Yu ◽  
Zhenhua Chen ◽  
Guanfeng Lin ◽  
Zhigao Zhang ◽  
...  

Aim: Development of a routine screening technique for Chlamydia trachomatis infection. The proposed approach involves the CRISPR RNA (crRNA). In the presence of the target sequence, the RNase activity of the Cas13a protein is activated, and it cleaves RNA fluorescent probe so that fluorescence will be emitted. Results: The sensitivity of the detection based on CRISPR-Cas13a was 10 fM. The results obtained by CRISPR-Cas13a and quantitative polymerase chain reaction were closely correlated: χ2 = 81.798 (p < 0.001). Conclusion: The method can be carried out at room temperature and yields results within 2 h. The developed technique does not require expensive instruments and, thus, can meet the needs of community hospitals and other institutions for screening.


2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.


2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.


Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 78
Author(s):  
Melissa Bello-Perez ◽  
Mikolaj Adamek ◽  
Julio Coll ◽  
Antonio Figueras ◽  
Beatriz Novoa ◽  
...  

Recent studies suggest that short pentraxins in fish might serve as biomarkers for not only bacterial infections, as in higher vertebrates including humans, but also for viral ones. These fish orthologs of mammalian short pentraxins are currently attracting interest because of their newly discovered antiviral activity. In the present work, the modulation of the gene expression of all zebrafish short pentraxins (CRP-like proteins, CRP1-7) was extensively analyzed by quantitative polymerase chain reaction. Initially, the tissue distribution of crp1-7 transcripts and how the transcripts varied in response to a bath infection with the spring viremia of carp virus, were determined. The expression of crp1-7 was widely distributed and generally increased after infection (mostly at 5 days post infection), except for crp1 (downregulated). Interestingly, several crp transcription levels significantly increased in skin. Further assays in mutant zebrafish of recombinant activation gene 1 (rag1) showed that all crps (except for crp2, downregulated) were already constitutively highly expressed in skin from rag1 knockouts and only increased moderately after viral infection. Similar results were obtained for most mx isoforms (a reporter gene of the interferon response), suggesting a general overcompensation of the innate immunity in the absence of the adaptive one.


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