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ABSTRACT Carryover contamination during amplicon sequencing workflow (AMP-Seq) put the accuracy of the high-throughput detection for pathogens at risk. The purpose of this study is to develop a carryover contaminations-controlled AMP-Seq (ccAMP-Seq) workflow to enable accurate qualitative and quantitative detection for pathogens. By using the AMP-Seq workflow to detect SARS-CoV-2, Aerosols, reagents and pipettes were identified as potential sources of contaminations and ccAMP-Seq was then developed. ccAMP-Seq used filter tips and physically isolation of experimental steps to avoid cross contamination, synthetic DNA spike-ins to compete with contaminations and quantify SARS-CoV-2, dUTP/uracil DNA glycosylase system to digest the carryover contaminations, and a new data analysis procedure to remove the sequencing reads from contaminations. Compared to AMP-Seq, the contamination level of ccAMP-Seq was at least 22-folds lower and the detection limit was also about an order of magnitude lower—as low as one copy/reaction. By testing the dilution series of SARS-CoV-2 nucleic acid standard, ccAMP-Seq showed 100% sensitivity and specificity. The high sensitivity of ccAMP-Seq was further confirmed by the detection of SARS-CoV-2 from 62 clinical samples. The consistency between qPCR and ccAMP-Seq was 100% for all the 53 qPCR-positive clinical samples. Seven qPCR-negative clinical samples were found to be positive by ccAMP-Seq, which was confirmed by extra qPCR tests on subsequent samples from the same patients. This study presents a carryover contamination-controlled, accurate qualitative and quantitative amplicon sequencing workflow that addresses the critical problem of pathogen detection for infectious diseases. IMPORTANCE Accuracy, a key indicator of pathogen detection technology, is compromised by carryover contamination in the amplicon sequencing workflow. Taking the detection of SARS-CoV-2 as case, this study presents a new carryover contamination-controlled amplicon sequencing workflow. The new workflow significantly reduces the degree of contamination in the workflow, thereby significantly improving the accuracy and sensitivity of the SARS-CoV-2 detection and empowering the ability of quantitative detection. More importantly, the use of the new workflow is simple and economical. Therefore, the results of this study can be easily applied to other microorganism, which has great significance for improving the detection level of microorganism.

ABSTRACT: Objectives: Preventing reverse transcription loop-mediated isothermal amplification (RT-LAMP) carryover contamination could be solved by adding deoxyuridine triphosphate (dUTP) and uracil-DNA glycosylase (UDG) into the reaction master mix. Methods: RNA was extracted from nasopharyngeal swab samples by a simple RNA extraction method. Results: Testing of 77 samples demonstrated 91.2% sensitivity (95% confidence interval [CI]: 78–98.2%) and 100% specificity (95% confidence interval: 92–100%) using UDG RT-LAMP. Conclusion: This colorimetric UDG RT-LAMP is a simple-to-use, fast, and easy-to-interpret method, which could serve as an alternative for diagnosis of SARS-CoV-2 infection, especially in remote hospitals and laboratories with under-equipped medical facilities.

Loop-mediated isothermal amplification (LAMP) is being used as a robust rapid diagnostic tool to prevent the transmission of infectious diseases. However, carryover contamination of LAMP-amplified products originating from previous tests has been a problem in LAMP-based bio-analytical assays. In this study, we developed a Cod-uracil-DNA-glycosylase real-time reverse transcriptase LAMP assay (Cod-UNG-rRT-LAMP) for the elimination of carryover contamination and the rapid detection of SARS-CoV-2 in point-of-care (POC) testing. Using the Cod-UNG-rRT-LAMP assay, the SARS-CoV-2 virus could be detected as low as 2 copies/µl (8 copies/reaction) within 45 min of amplification and 2.63 ± 0.17 pg (equivalent to 2.296 × 109 copies) of contaminants per reaction could be eliminated. Analysis of clinical SARS-CoV-2 samples using the Cod-UNG-rRT-LAMP assay showed an excellent agreement with a relative accuracy of 98.2%, sensitivity of 97.1%, and specificity of 95.2% in comparison to rRT-PCR. The results obtained in this study clearly demonstrate the feasibility of the use of the Cod-UNG-rRT-LAMP assay for applications toward the POC diagnosis of SARS-CoV-2 and on-site testing of other pathogens. [ABSTRACT FROM AUTHOR]

Loop-mediated isothermal amplification (LAMP) is being used as a robust rapid diagnostic tool to prevent the transmission of infectious diseases. However, carryover contamination of LAMP-amplified products originating from previous tests has been a problem in LAMP-based bio-analytical assays. In this study, we developed a Cod-uracil-DNA-glycosylase real-time reverse transcriptase LAMP assay (Cod-UNG-rRT-LAMP) for the elimination of carryover contamination and the rapid detection of SARS-CoV-2 in point-of-care (POC) testing. Using the Cod-UNG-rRT-LAMP assay, the SARS-CoV-2 virus could be detected as low as 2 copies/µl (8 copies/reaction) within 45 min of amplification and 2.63 ± 0.17 pg (equivalent to 2.296 × 109 copies) of contaminants per reaction could be eliminated. Analysis of clinical SARS-CoV-2 samples using the Cod-UNG-rRT-LAMP assay showed an excellent agreement with a relative accuracy of 98.2%, sensitivity of 97.1%, and specificity of 95.2% in comparison to rRT-PCR. The results obtained in this study clearly demonstrate the feasibility of the use of the Cod-UNG-rRT-LAMP assay for applications toward the POC diagnosis of SARS-CoV-2 and on-site testing of other pathogens.

• RNA-purification–free method was developed for the detection of SARS-CoV-2 • One-step uracil-DNA glycosylase reverse transcription loop-mediated isothermal amplification was used to detect SARS-CoV-2 • Result were obtained by direct observation of end products by the naked eye Preventing reverse transcription loop-mediated isothermal amplification (RT-LAMP) carryover contamination could be solved by adding deoxyuridine triphosphate (dUTP) and uracil-DNA glycosylase (UDG) into the reaction master mix. RNA was extracted from nasopharyngeal swab samples by a simple RNA extraction method. Testing of 77 samples demonstrated 91.2% sensitivity (95% confidence interval [CI]: 78–98.2%) and 100% specificity (95% confidence interval: 92–100%) using UDG RT-LAMP. This colorimetric UDG RT-LAMP is a simple-to-use, fast, and easy-to-interpret method, which could serve as an alternative for diagnosis of SARS-CoV-2 infection, especially in remote hospitals and laboratories with under-equipped medical facilities. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae is an important clinical pathogenic bacterium that is the primary cause of meningitis, septicemia and community-acquired pneumonia. The mortality rate of pneumococcal disease is high, especially in children younger than 5-years-old. Rapid and accurate detection of S. pneumoniae is critical for clinical diagnosis. A ply gene-based multiple cross displacement amplification (MCDA) assay, amplifying DNA under 65°C for 40 min, was established to detect S. pneumoniae. Antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) was applied to prevent carryover contamination. A lateral flow biosensor (LFB) was used to indicate the MCDA results. The ply -MCDA assay could detect as low as 10 fg of S. pneumoniae DNA and 447 colony forming units (CFU)/mL of spiked sputum samples. The analytical sensitivity of the ply -MCDA assay to detect clinical specimens was 100 times higher than that of PCR. The specificity of the ply -MCDA assay was evaluated using 15 S. pneumoniae strains and 25 non- S. pneumoniae strains, which confirmed the high selectivity of the ply -MCDA assay for S. pneumoniae. The AUDG enzyme could effectively eliminate carryover contamination and thus prevented false-positive results. In conclusion, ply -AUDG-MCDA-LFB is a simple, rapid and accurate method to detect S. pneumoniae. [ABSTRACT FROM AUTHOR]

The current report devised a novel isothermal diagnostic assay, termed as nanoparticle-based biosensor (NB)- and antarctic thermal sensitive uracil-DNA-glycosylase (ATSU)-supplemented polymerase spiral reaction (PSR; NB-ATSU-PSR). The technique merges enzymatic digestion of carryover contaminants and isothermal nucleic acid amplification technique (PSR) for simultaneous detection of nucleic acid sequences and elimination of carryover contamination. In particular, nucleic acid amplification and elimination of carryover contamination are conducted in a single pot and, thus, the use of a closed-tube reaction can remove undesired results due to carryover contamination. For demonstration purpose, Klebsiella pneumoniae is employed as the model to demonstrate the usability of NB-ATSU-PSR assay. The assay's sensitivity, specificity, and practical feasibility were successfully evaluated using the pure cultures and sputum samples. The amplification products were detectable from as little as 100 fg of genomic DNAs and from ~550 colony-forming unit (CFU) in 1 ml of spiked sputum samples. All K. pneumoniae strains examined were positive for NB-ATSU-PSR detection, and all non-K. pneumoniae strains tested were negative for the NB-ATSU-PSR technique. The whole process, including rapid template preparation (20 min), PSR amplification (60 min), ATSU treatment (5 min), and result reporting (within 2 min), can be finished within 90 min. As a proof-of-concept methodology, NB-ATSU-PSR technique can be reconfigured to detect various target nucleic acid sequences by redesigning the PSR primer set.

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In the present study, IS 6110 loop mediated isothermal amplification (LAMP) assay was modified using dUTP-UNG (uracil-DNA N -glycosylase) strategy to prevent carryover contamination, and was evaluated using clinical specimens. The clinical specimens were collected from 236 suspected patients of pulmonary tuberculosis and 315 specimens of suspected patients of extra pulmonary tuberculosis. DNA was extracted from specimens and used as template for nucleic acid amplification. The results were evaluated with culture method as gold standard. Modified IS 6110 LAMP assay showed high sensitivity (94.4%) and specificity (97.2%) in specimens collected from suspected pulmonary tuberculosis patients. Sensitivity was comparatively less (86.67%) in extra pulmonary specimens while specificity was 94.04%. In conclusion, IS 6110 LAMP assay was modified to prevent carry over contamination and it was validated to be rapid, sensitive and specific method with prospective application in resource-limited settings. [ABSTRACT FROM AUTHOR]

Carryover contamination during amplicon sequencing workflow (AMP-Seq) put the accuracy of the high-throughput detection for pathogens at risk. The purpose of this study is to develop a carryover contaminations-controlled AMP-Seq (ccAMP-Seq) workflow to enable accurate qualitative and quantitative detection for pathogens. By using the AMP-Seq workflow to detect SARS-CoV-2, Aerosols, reagents and pipettes were identified as potential sources of contaminations and ccAMP-Seq was then developed. ccAMP-Seq used filter tips and physically isolation of experimental steps to avoid cross contamination, synthetic DNA spike-ins to compete with contaminations and quantify SARS-CoV-2, dUTP/uracil DNA glycosylase system to digest the carryover contaminations, and a new data analysis procedure to remove the sequencing reads from contaminations. Compared to AMP-Seq, the contamination level of ccAMP-Seq was at least 22-folds lower and the detection limit was also about an order of magnitude lower-as low as one copy/reaction. By testing the dilution series of SARS-CoV-2 nucleic acid standard, ccAMP-Seq showed 100% sensitivity and specificity. The high sensitivity of ccAMP-Seq was further confirmed by the detection of SARS-CoV-2 from 62 clinical samples. The consistency between qPCR and ccAMP-Seq was 100% for all the 53 qPCR-positive clinical samples. Seven qPCR-negative clinical samples were found to be positive by ccAMP-Seq, which was confirmed by extra qPCR tests on subsequent samples from the same patients. This study presents a carryover contamination-controlled, accurate qualitative and quantitative amplicon sequencing workflow that addresses the critical problem of pathogen detection for infectious diseases. IMPORTANCE Accuracy, a key indicator of pathogen detection technology, is compromised by carryover contamination in the amplicon sequencing workflow. Taking the detection of SARS-CoV-2 as case, this study presents a new carryover contamination-controlled amplicon sequencing workflow. The new workflow significantly reduces the degree of contamination in the workflow, thereby significantly improving the accuracy and sensitivity of the SARS-CoV-2 detection and empowering the ability of quantitative detection. More importantly, the use of the new workflow is simple and economical. Therefore, the results of this study can be easily applied to other microorganism, which has great significance for improving the detection level of microorganism., Competing Interests: The authors declare no conflict of interest.

Conventional and routine diagnostics such as polymerase chain reaction (PCR) and serological tests are less sensitive, costly, and require sample pretreatment procedures. CRISPR/Cas systems that inherently assist bacteria and archaea in destroying invading phage genetic materials via an RNA-mediated interference strategy have been reconstituted in vitro and harnessed for nucleic and non-nucleic acid diagnostics. CRISPR/Cas-based diagnostics (CRISPR-Dx) are cost-effective, possess excellent sensitivity (attomolar) and specificity (single base distinction), exhibit fast turnaround response, and support nucleic acid extraction-free workflow. However, CRISPR-Dx still needs to address various challenges to translate the laboratory work into end-user tailored solutions. In this perspective, we review the relevant progress of CRISPR/Cas systems-based diagnostics, focusing on the comprehensive customization and applications of leading and trending CRISPR/Cas systems as platform technologies for fluorescence, colorimetric, and electrical signal detection. The impact of the CRISPR game-changing technology on the COVID-19 pandemic is highlighted. We also demonstrate the role of CRISPR/Cas systems for carryover contamination prevention. The advancements in signal amplification strategies using engineered crRNAs, novel reporters, nanoparticles, artificial genetic circuits, microfluidics, and smartphones are also covered. Furthermore, we critically discuss the translation of CRISPR-Dx's basic research into end-user diagnostics for commercialization success in the near future. Finally, we discuss the complex challenges and alternative solutions to harness the CRISPR/Cas potential in detail. Broad applications of CRISPR Cas systems. CRISPR technology is dedicated to the transformation of biological sciences, biotechnology, food industry, and medicine. [Display omitted] • The comprehensive customization and applications of the leading and trending CRISPR/Cas systems are discussed. • The catalytic behavior of Cas nucleases on engineered sg/crRNA and novel reporters extend the general applicability of CRISPR/Cas systems. • The rational programming of CRISPR/Cas systems prevent amplification carryover contamination. • Nanoparticles, artificial genetic circuits, microfluidics, and smartphones advance the CRISPR/Cas systems signal amplification strategies. • CRISPR/Cas-based diagnostics aimed at fulfilling the ASSURED criteria could pave the way for commercialization success in the near future. [ABSTRACT FROM AUTHOR]

Abstract: Owing to its selective uracil-excision property, uracil-DNA glycosylase (UDG) has been widely utilized in diagnostic PCR applications as an effective decontamination method. Since mesophilic UDGs in PCR has been shown to degrade not just contaminant DNA but also target amplicon, there has been an increase in demand for cold-active UDGs. We characterized UDG from Photobacterium aplysiae GMD509 (Pap GMD509 UDG) expressed in Escherichia coli BL21 (DE3). The optimal temperature range of the enzyme was 25–30°C, which is considerably lower than any other reported UDG, and the half-life of the enzyme at 40°C and 50°C was approximately 77s and 33s, respectively. These results clearly demonstrate the fragility of this enzyme upon heating. In addition, we compared the carryover contamination control property of Pap GMD509 UDG with other commercialized UDGs. The results indicate that Pap GMD509 UDG is capable of degrading contaminant DNA without a preincubation step before the main PCR reaction. These attributes imply that the Pap GMD509 UDG is a highly adequate enzyme to prevent carryover contamination during PCR. [Copyright &y& Elsevier]

The current report devised a novel isothermal diagnostic assay, termed as nanoparticle-based biosensor (NB)- and antarctic thermal sensitive uracil-DNA-glycosylase (ATSU)-supplemented polymerase spiral reaction (PSR; NB-ATSU-PSR). The technique merges enzymatic digestion of carryover contaminants and isothermal nucleic acid amplification technique (PSR) for simultaneous detection of nucleic acid sequences and elimination of carryover contamination. In particular, nucleic acid amplification and elimination of carryover contamination are conducted in a single pot and, thus, the use of a closed-tube reaction can remove undesired results due to carryover contamination. For demonstration purpose, Klebsiella pneumoniae is employed as the model to demonstrate the usability of NB-ATSU-PSR assay. The assay's sensitivity, specificity, and practical feasibility were successfully evaluated using the pure cultures and sputum samples. The amplification products were detectable from as little as 100 fg of genomic DNAs and from ~550 colony-forming unit (CFU) in 1 ml of spiked sputum samples. All K. pneumoniae strains examined were positive for NB-ATSU-PSR detection, and all non- K. pneumoniae strains tested were negative for the NB-ATSU-PSR technique. The whole process, including rapid template preparation (20 min), PSR amplification (60 min), ATSU treatment (5 min), and result reporting (within 2 min), can be finished within 90 min. As a proof-of-concept methodology, NB-ATSU-PSR technique can be reconfigured to detect various target nucleic acid sequences by redesigning the PSR primer set., (Copyright © 2019 Wang, Jiao, Wang, Sun, Li, Wang, Xiao, Shen, Xu, Qi, Wang, Guo and Shen.)

A letter to the editor is presented in response to the article on the risk of false positive hepatitis C virus RNA due to sample carryover on an automated hematology analyzer.