scholarly journals An integrated lab-on-a-chip device for RNA extraction, amplification and CRISPR-Cas12a-assisted detection for COVID-19 screening in resource-limited settings

2022 ◽  
Author(s):  
Bongkot Ngamsom ◽  
Alexander Iles ◽  
Moses Kamita ◽  
Racheal Kimani ◽  
Pablo Rodriguez-Mateos ◽  
...  
Keyword(s):  
Saliva Sample ◽  
Rna Extraction ◽  
Lab On A Chip ◽  
Cross Reactivity ◽  
Contact Tracing ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Sample Collection ◽  
Middle Income ◽  
Specificity And Sensitivity

In response to the ongoing COVID-19 pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip platform, namely IFAST-CRISPR, as an affordable, rapid and high-precision molecular diagnostic means for SARS-CoV-2 detection. The herein proposed sample-to-answer platform integrates RNA extraction, amplification and CRISPR-Cas-based detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and GuHCl, streamline sample preparation, including RNA concentration, extraction and purification, in 15 min with minimal hands-on steps. By combining RT-LAMP with CRISPR-Cas12 assays targeting the nucleoprotein (N) gene, visual identification of ≥ 470 copies mL-1 genomic SARS-CoV-2 samples was achieved in 45 min, with no cross-reactivity towards HCoV-OC43 nor H1N1. On-chip assays showed the ability to isolate and detect SARS-CoV-2 from 1,000 genome copies mL-1 of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity and sensitivity-comparable alternative to the costly gold-standard RT-PCR assay, requiring only a simple heating source. Further investigations on multiplexing and direct interfacing of the accessible Swan-brand cigarette filter for saliva sample collection could provide a complete work flow for COVID-19 diagnostics from saliva samples suitable for low-resource settings.

scholarly journals Clinical validation of colorimetric RT-LAMP, a fast, highly sensitive and specific COVID-19 molecular diagnostic tool that is robust to detect SARS-CoV-2 variants of concern

2021 ◽  
Author(s):  
Pedro Augusto Alves ◽  
Ellen G. Oliveira ◽  
Ana Paula M. Franco-Luiz ◽  
Leticia T. Almeida ◽  
Amanda B. Goncalves ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Rna Extraction ◽  
High Specificity ◽  
Cross Reactivity ◽  
Alternative Methods ◽  
Molecular Diagnostic ◽  
N Gene ◽  
Clinical Validation ◽  
Rna Detection ◽  
Viral Transport Medium

The COVID-19 pandemics unfolded due to the widespread SARS-CoV-2 transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric RT-LAMP (reverse transcription loop-mediated isothermal amplification) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 viral genomic copies/uL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65 C for 30 min. When compared to RT-qPCR, up to Ct value 32, RT-LAMP presented 97% (87.4-99.4% 95% CI) sensitivity and 100% (86.2-100%) specificity for SARS-CoV-2 RNA detection targeting N gene. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern (VOC) and variants of interest (VOI), such as variants occurring in Brazil named P.1, P.2, B.1.1.374 and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipments, infrastructure and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

scholarly journals Extraction-free rapid cycle RT-qPCR and extreme RT-PCR for SARS-CoV-2 virus detection

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S139-S139
Author(s):  
J C Lownik ◽  
J S Farrar ◽  
G Way ◽  
R K Martin
Keyword(s):  
Supply Chain ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Virus Detection ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Sample Collection ◽  
Rt Pcr ◽  
Hydrolysis Probe ◽  
Time Requirements

Abstract Introduction/Objective Since the start of the coronavirus disease 2019 (COVID-19) pandemic, molecular diagnostic testing for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has faced substantial supply chain shortages and noteworthy delays in result reporting after sample collection. Supply chain shortages have been most evident in reagents for RNA extraction and rapid diagnostic testing. In this study, we explored the kinetic limitations of extraction-free rapid cycle RT-qPCR for SARS-CoV-2 virus detection using the commercially available capillary based LightCycler. Methods/Case Report We optimized reverse transcription and PCR under extraction-free and rapid thermocycling conditions utilizing hydrolysis probe-based detection methods using a Roche LightCycler. Results (if a Case Study enter NA) This protocol improves detection speed while maintaining the sensitivity and specificity of hydrolysis probe-based detection. Percentage agreement between the developed assay and previously tested positive patient samples was 97.6% (n= 40/41) and negative patient samples was 100% (40/40). We further demonstrate that using purified RNA, SARS-CoV-2 testing using extreme RT-PCR and product verification by melting can be completed in less than 3 minutes. Conclusion We developed a protocol for sensitive and specific RT-qPCR of SARS-CoV-2 RNA from nasopharyngeal swabs in less than 20 minutes, with minimal hands-on time requirements. Overall, these studies provide a framework for increasing the speed of SARS-CoV-2 and other infectious disease testing.

scholarly journals Patient DNA cross-reactivity of the CDC SARS-CoV-2 extraction control leads to an inherent potential for false negative results

2020 ◽  
Author(s):  
Adam P. Rosebrock
Keyword(s):  
Reverse Transcription ◽  
False Negative ◽  
Rna Extraction ◽  
Cross Reactivity ◽  
Sample Collection ◽  
Negative Results ◽  
False Negative Results ◽  
Important Diagnostic Tool ◽  
Control And Prevention ◽  
Careful Handling

AbstractTesting for RNA viruses such as SARS-CoV-2 requires careful handling of inherently labile RNA during sample collection, clinical processing, and molecular analysis. Tests must include fail-safe controls that affirmatively report the presence of intact RNA and demonstrate success of all steps of the assay. A result of “no virus signal” is insufficient for clinical interpretation: controls must also say “The reaction worked as intended and would have found virus if present.” Unfortunately, a widely used test specified by the US Centers for Disease Control and Prevention (CDC) incorporates a control that does not perform as intended and claimed. Detecting SARS-CoV-2 with this assay requires both intact RNA and successful reverse transcription. The CDC-specified control does not require either of these, due to its inability to differentiate human genomic DNA from reverse-transcribed RNA. Patient DNA is copurified from nasopharyngeal swabs during clinically-approved RNA extraction and is sufficient to return an “extraction control success” signal using the CDC design. As such, this assay fails-unsafe: truly positive patient samples return a false-negative result of “no virus detected, control succeeded” following any of several readily-encountered mishaps. This problem affects tens-of-millions of patients worth of shipped assays, but many of these flawed reagents have not yet been used. There is an opportunity to improve this important diagnostic tool. As demonstrated here, a re-designed transcript-specific control correctly monitors sample collection, extraction, reverse transcription, and qPCR detection. This approach can be rapidly implemented and will help reduce truly positive patients from being incorrectly given the all-clear.One Sentence SummaryA widely-used COVID-19 diagnostic is mis-designed and generates false-negative results, dangerously confusing “No” with “Don’t know” – but it’s fixable

scholarly journals Prospective Comparison of Saliva and Nasopharyngeal Swab Sampling for Mass Screening for COVID-19

2021 ◽  
Vol 8 ◽  
Author(s):  
Mathieu Nacher ◽  
Mayka Mergeay-Fabre ◽  
Denis Blanchet ◽  
Orelie Benoit ◽  
Tristan Pozl ◽  
...  
Keyword(s):  
Mass Screening ◽  
Saliva Sample ◽  
Field Testing ◽  
Contact Tracing ◽  
N Gene ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Prospective Comparison ◽  
Asymptomatic Patients ◽  
Patient Groups

Current testing for COVID-19 relies on reverse-transcriptase polymerase chain reaction from a nasopharyngeal swab specimen. Saliva samples have advantages regarding ease and painlessness of collection, which does not require trained staff and may allow self-sampling. We enrolled 776 persons at various field-testing sites and collected nasopharyngeal and pooled saliva samples. One hundred sixty two had a positive COVID-19 RT-PCR, 61% were mildly symptomatic and 39% asymptomatic. The sensitivity of RT-PCR on saliva samples vs. nasopharygeal swabs varied depending on the patient groups considered or on Ct thresholds. There were 10 (6.2%) patients with a positive saliva sample and a negative nasopharyngeal swab, all of whom had Ct values <25 for three genes. For symptomatic patients for whom the interval between symptoms onset and sampling was <10 days sensitivity was 77% but when excluding persons with isolated N gene positivity (54/162), sensitivity was 90%. In asymptomatic patients, the sensitivity was only 24%. When we looked at patients with Cts <30, sensitivity was 83 or 88.9% when considering two genes. The relatively good performance for patients with low Cts suggests that Saliva testing could be a useful and acceptable tool to identify infectious persons in mass screening contexts, a strategically important task for contact tracing and isolation in the community.

Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

2020 ◽  
Vol 15 (15) ◽  
pp. 1483-1487
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Allan Njau ◽  
Sudha Ananth ◽  
Kimya Jones ◽  
...  
Keyword(s):  
Supply Chain ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Sample Collection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Short Supply ◽  
Viral Transport ◽  
3D Printed ◽  
Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

scholarly journals COVID-19 Antibody Detecting Rapid Diagnostic Tests Show High Cross-Reactivity When Challenged with Pre-Pandemic Malaria, Schistosomiasis and Dengue Samples

Diagnostics ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1163
Author(s):  
Fien Vanroye ◽  
Dorien Van den Bossche ◽  
Isabel Brosius ◽  
Bieke Tack ◽  
Marjan Van Esbroeck ◽  
...  
Keyword(s):  
Retrospective Study ◽  
Whole Blood ◽  
Diagnostic Tests ◽  
Risk Mitigation ◽  
Plasmodium Species ◽  
Cross Reactivity ◽  
Rapid Diagnostic Tests ◽  
Middle Income ◽  
Middle Income Countries ◽  
Independent Evaluation

COVID-19 Antibody Detecting Rapid Diagnostic Tests (COVID-19 Ab RDTs) are the preferred tool for SARS-CoV-2 seroprevalence studies, particularly in low- and middle-income countries. The present study challenged COVID-19 Ab RDTs with pre-pandemic samples of patients exposed to tropical pathogens. A retrospective study was performed on archived serum (n = 94) and EDTA whole blood (n = 126) samples obtained during 2010–2018 from 196 travelers with malaria (n = 170), schistosomiasis (n = 25) and dengue (n = 25). COVID-19 Ab RDTs were selected based on regulatory approval status, independent evaluation results and detecting antigens. Among 13 COVID-19 Ab RDT products, overall cross-reactivity was 18.5%; cross-reactivity for malaria, schistosomiasis and dengue was 20.3%, 18.1% and 7.5%, respectively. Cross-reactivity for current and recent malaria, malaria antibodies, Plasmodium species and parasite densities was similar. Cross-reactivity among the different RDT products ranged from 2.7% to 48.9% (median value 14.5%). IgM represented 67.9% of cross-reactive test lines. Cross-reactivity was not associated with detecting antigens, patient categories or disease (sub)groups, except for schistosomiasis (two products with ≥60% cross-reactivity). The high cross-reactivity for malaria, schistosomiasis and—to a lesser extent—dengue calls for risk mitigation when using COVID-19 Ab RDTs in co-endemic regions.

scholarly journals Autoimmune Responses in Oncology: Causes and Significance

2021 ◽  
Vol 22 (15) ◽  
pp. 8030
Author(s):  
Halin Bareke ◽  
Pablo Juanes-Velasco ◽  
Alicia Landeira-Viñuela ◽  
Angela-Patricia Hernandez ◽  
Juan Jesús Cruz ◽  
...  
Keyword(s):  
Cancer Immunotherapy ◽  
Protein Microarrays ◽  
Cross Reactivity ◽  
Fine Tuning ◽  
Cancer Treatments ◽  
Aberrant Expression ◽  
Specificity And Sensitivity ◽  
Anti Cancer ◽  
Life Threatening ◽  
Considerable Impact

Specific anti-tumor immune responses have proven to be pivotal in shaping tumorigenesis and tumor progression in solid cancers. These responses can also be of an autoimmune nature, and autoantibodies can sometimes be present even before the onset of clinically overt disease. Autoantibodies can be generated due to mutated gene products, aberrant expression and post-transcriptional modification of proteins, a pro-immunogenic milieu, anti-cancer treatments, cross-reactivity of tumor-specific lymphocytes, epitope spreading, and microbiota-related and genetic factors. Understanding these responses has implications for both basic and clinical immunology. Autoantibodies in solid cancers can be used for early detection of cancer as well as for biomarkers of prognosis and treatment response. High-throughput techniques such as protein microarrays make parallel detection of multiple autoantibodies for increased specificity and sensitivity feasible, affordable, and quick. Cancer immunotherapy has revolutionized cancer treatments and has made a considerable impact on reducing cancer-associated morbidity and mortality. However, immunotherapeutic interventions such as immune checkpoint inhibition can induce immune-related toxicities, which can even be life-threatening. Uncovering the reasons for treatment-induced autoimmunity can lead to fine-tuning of cancer immunotherapy approaches to evade toxic events while inducing an effective anti-tumor immune response.

Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV

2021 ◽  
Author(s):  
Liyun Chang ◽  
Zhiyong Liu ◽  
Yuelan Zhao ◽  
Yan Li ◽  
Jianhua Qin
Keyword(s):  
Quantitative Pcr ◽  
Quantitative Methods ◽  
Diarrheal Disease ◽  
Quantitative Polymerase Chain Reaction ◽  
Simultaneous Detection ◽  
Cross Reactivity ◽  
N Gene ◽  
Established Method ◽  
Diarrhea Virus ◽  
Fluorescence Quantitative Pcr

Background: In this study, we aimed to establish a multiplex fluorescence quantitative polymerase chain reaction (PCR) method for the identification and detection of bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV). Methods: Based on the highly conserved sequences of BVDV E2 gene, BRV VP6 gene and BCV N gene in GenBank, specific primers were designed to amplify the target gene fragments of each virus and the reaction conditions and system were optimized. Multiple fluorescence quantitative methods were established by fluorescence quantitative PCR. Result: The minimum detection limits of plasmid standards for BVDV, BRV and BCV by multiplex fluorescence quantitative PCR were 1.19×102 copies/μL, 3.89×101 copies/μL and 3.74×101 copies/μL, respectively. The lowest sensitivity of the established method was 100 times higher than that of conventional PCR and had high sensitivity. Furthermore, BVDV, BRV and BCV were amplified specifically, with no cross-reactivity with Escherichia coli (E. coli), Salmonella and infectious bovine rhinotracheitis virus (IBRV). The intra-and inter-group coefficients of variation were less than 1%, showing good assay repeatability. Using the established method and ordinary multiplex PCR to simultaneously detect 150 clinical diarrheal disease material samples, the coincidence rate of samples with mixed infection of the three viruses was 83.3%. The results showed that the multiplex fluorescent quantitative PCR detection method established in this study provides a rapid, sensitive and specific technique for clinical diagnosis and epidemiological monitoring of BVDV, BRV and BCV.

scholarly journals One-step RNA extraction for RT-qPCR detection of 2019-nCoV

2020 ◽  
Author(s):  
Monica Sentmanat ◽  
Evguenia Kouranova ◽  
Xiaoxia Cui
Keyword(s):  
Real Time ◽  
Rna Extraction ◽  
Virus Transmission ◽  
Extraction Methods ◽  
Viral Rna ◽  
Taqman Probe ◽  
N Gene ◽  
Structural Protein ◽  
Diagnostic Panel ◽  
Primer Sets

ABSTRACTThe global outbreak of coronavirus disease 2019 (COVID-19) has placed an unprecedented burden on healthcare systems as the virus spread from the initial 27 reported cases in the city of Wuhan, China to a global pandemic in under three month[1]. Resources essential to monitoring virus transmission have been challenged with a demand for expanded surveillance. The CDC 2019-nCoV Real-Time Diagnostic Panel uses a real-time reverse transcription polymerase chain reaction (RT-PCR) consisting of two TaqMan probe and primer sets specific for the 2019-nCoV N gene, which codes for the nucleocapsid structural protein that encapsulates viral RNA, for the qualitative detection of 2019-nCoV viral RNA in respiratory samples. To isolate RNA from respiratory samples, the CDC lists RNA extraction kits from four manufacturers. In anticipation of a limited supply chain of RNA extraction kits and the need for test scalability, we sought to identify alternative RNA extraction methods. Here we show that direct lysis of respiratory samples can be used in place of RNA extraction kits to run the CDC 2019-nCoV Real-Time Diagnostic assay with the additional benefits of higher throughput, lower cost, faster turnaround and possibly higher sensitivity and improved safety.

scholarly journals Molecular diagnostic investigation of brucellosis in a caprine organic farm

2017 ◽  
Vol 57 (3) ◽  
pp. 217
Author(s):  
J. A. IKONOMOPOULOS (I.A. ΟΙΚΟΝΟΜΟΠΟΥΛΟΣ) ◽  
M. GAZOULI (M. ΓΑΖΟΥΛΗ) ◽  
E. XYLOURI (Ε. ΞΥΛΟΥΡΗ) ◽  
E. K. GEORGAKILAS (E.K. ΓΕΩΡΓΑΚΙΛΑΣ) ◽  
P. KARAGIANNI (Π. ΚΑΡΑΓΙΑΝΝΗ) ◽  
...  
Keyword(s):  
Brucella Melitensis ◽  
Molecular Diagnostic ◽  
Blood Collection ◽  
Sample Collection ◽  
Diagnostic Investigation ◽  
Microbial Identification ◽  
Organic Farm ◽  
Serious Disease ◽  
Public Health Protection ◽  
First Time

Brucellosis of sheep and goats is widely spread in the Mediterranean basin. The disease is of considerable significance with connection to Public Health protection since it can be transmitted to humans causing serious disease. In April 2005 we investigated whether brucellosis was present among the male animals of a caprine organic herd in Nomos Hleias, Greece. The herd consisted of 250 female and 13 male animals and had a record of sporadic abortions usually taking place at the final third of gestation. During sample collection all the animals were found clinically healthy, although it was not possible to determine from the records of the farm if there was any previous incidence of orchitis or epididymitis in the animals under study. The laboratory diagnostic investigation consisted of a polymerase chain reaction (PCR) assay applied for the detection of DNA belonging to Brucella melitensis in blood samples. Two of the 13 samples that were tested reacted positive by PCR allowing the amplification of the 844 base pair DNA fragment specific for Brucella melitensis. In this study, PCR allowed detection of two animals that although they were clinically healthy, they carried enough Brucella in their blood to allow microbial identification with only a single blood collection. This finding seems to agree with the concept that goats consist the reservoir of brucellosis in Greece developing milder disease than sheep and sustaining the infection for longer periods of time. One of the positive samples that were recorded was identified by sequencing as Brucella suis, something that is reported for the first time with connection to goats. This finding and the sensitivity of man to B. suis renders human exposure to this pathogen through goats, an epidemiology factor worth of detailed investigation. This necessity is associated with the fact that as opposed to porcine meat, sheep and goat dairy products in Greece are sometimes consumed without the necessary heat treatment.

Sign in / Sign up

Export Citation Format

Share Document