scholarly journals RT-LAMP CRISPR-Cas12/13-Based SARS-CoV-2 Detection Methods

Diagnostics ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1646
Author(s):  
Kasturi Selvam ◽  
Mohamad Ahmad Najib ◽  
Muhammad Fazli Khalid ◽  
Suharni Mohamad ◽  
Fahreddin Palaz ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Public Attention ◽  
Laboratory Equipment ◽  
Highly Sensitive ◽  
Polymerase Chain

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has attracted public attention. The gold standard for diagnosing COVID-19 is reverse transcription–quantitative polymerase chain reaction (RT-qPCR). However, RT-qPCR can only be performed in centralized laboratories due to the requirement for advanced laboratory equipment and qualified workers. In the last decade, clustered regularly interspaced short palindromic repeats (CRISPR) technology has shown considerable promise in the development of rapid, highly sensitive, and specific molecular diagnostic methods that do not require complicated instrumentation. During the current COVID-19 pandemic, there has been growing interest in using CRISPR-based diagnostic techniques to develop rapid and accurate assays for detecting SARS-CoV-2. In this work, we review and summarize reverse-transcription loop-mediated isothermal amplification (RT-LAMP) CRISPR-based diagnostic techniques for detecting SARS-CoV-2.

scholarly journals Nucleic Acid Testing of SARS-CoV-2

2021 ◽  
Vol 22 (11) ◽  
pp. 6150
Author(s):  
Hee-Min Yoo ◽  
Il-Hwan Kim ◽  
Seil Kim
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Reverse Transcription ◽  
Diagnostic Methods ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Nucleic Acid Testing ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.

scholarly journals Review on Molecular Diagnosis of Cestode and Metacestode in Cattle

2021 ◽  
Vol 6 (1) ◽  
pp. 6-12
Author(s):  
Ziyad M. Bilal ◽  
◽  
Kedir S. Musa ◽  
Keyword(s):  
Sensitivity And Specificity ◽  
Diagnostic Techniques ◽  
Laboratory Research ◽  
Molecular Diagnostic ◽  
Enzyme Linked Immunosorbent Assay ◽  
Host Interaction ◽  
Highly Sensitive ◽  
Elisa Technique ◽  
Polymerase Chain ◽  
Higher Sensitivity

Cestode infestations in animals are the most important parasite of livestock and humans because most of these parasites are zoonotic causing cysticercosis and hydatidosis in man and it causes economic and production losses in livestock. Diagnosis of Taenia Spp by microscopic observation lack sensitivity and specificity and detection by enzyme-linked immunosorbent assay (ELISA) technique form cross-reaction. The molecular diagnostic can be best to detect in adult and larval stage in definitive and intermediate host based on the amplification of deoxyribonucleic acid (DNA) of target gene with the primer using a different technique of polymerase chain reaction (PCR) such as multiplex PCR. Conventional PCR, real-time PCR, nested PCR, and PCR-restriction fragment length polymorphism (RFLP) are highly sensitive for the diagnosis of cestode and metacestode. Those diagnoses are used for differentiation of Taenia species and differentiation of Taenia and Echinococcus species. As compared to other diagnostic techniques most molecular methods have higher sensitivity and specificity but due to the relatively higher cost, few are commercially available. Most of the molecular diagnostic tests developed to date are generally applicable for laboratory research purposes. The developments in the genomic and proteomic analysis should be used for further understanding of parasite-animal host interaction to find additional targets for diagnosis.

scholarly journals A Recent Update on Advanced Molecular Diagnostic Techniques for COVID-19 Pandemic: An Overview

2021 ◽  
Vol 12 ◽  
Author(s):  
Akanksha Roberts ◽  
Raghuraj Singh Chouhan ◽  
Deepshikha Shahdeo ◽  
Narlawar Sagar Shrikrishna ◽  
Veerbhan Kesarwani ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Electrochemical Sensors ◽  
Cost Effective ◽  
Surface Enhanced Raman Spectroscopy ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Techniques ◽  
Rt Pcr ◽  
Ongoing Research

Coronavirus disease 2019 (COVID-19), which started out as an outbreak of pneumonia, has now turned into a pandemic due to its rapid transmission. Besides developing a vaccine, rapid, accurate, and cost-effective diagnosis is essential for monitoring and combating the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its related variants on time with precision and accuracy. Currently, the gold standard for detection of SARS-CoV-2 is Reverse Transcription Polymerase Chain Reaction (RT-PCR), but it lacks accuracy, is time-consuming and cumbersome, and fails to detect multi-variant forms of the virus. Herein, we have summarized conventional diagnostic methods such as Chest-CT (Computed Tomography), RT-PCR, Loop Mediated Isothermal Amplification (LAMP), Reverse Transcription-LAMP (RT-LAMP), as well new modern diagnostics such as CRISPR–Cas-based assays, Surface Enhanced Raman Spectroscopy (SERS), Lateral Flow Assays (LFA), Graphene-Field Effect Transistor (GraFET), electrochemical sensors, immunosensors, antisense oligonucleotides (ASOs)-based assays, and microarrays for SARS-CoV-2 detection. This review will also provide an insight into an ongoing research and the possibility of developing more economical tools to tackle the COVID-19 pandemic.

scholarly journals Multiple-Centre Clinical Evaluation of Rapid Recombinase-Aided Amplification Assays for Five Pathogens

2021 ◽  
Author(s):  
Xin-xin Shen ◽  
Dan-wen Nie ◽  
Hong Zhang ◽  
Zhi-fei Zhan ◽  
Yuan Gao ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Clinical Performance ◽  
Quantitative Polymerase Chain Reaction ◽  
Human Adenovirus ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Polymerase Chain

Abstract Background: Recombinase-aided amplification(RAA) is a new, simple, and ultrafast isothermal molecular diagnostic technique performed within 30min at 39°C–42°C.In this study, we evaluated the clinical performance of four duplex RAA kits for hepatitis B virus(HBV), human adenovirus 3(HAdV3), human adenovirus 7(HAdV7), and Bordetella pertussis and one duplex reverse-transcription RAA (RT-RAA) kit for respiratory syncytial virus (RSV).Methods: A total of 392 sera and 374 respiratory tract samples were collected from five institutions in four China regions. Each RAA kit’s sensitivity and specificity were compared with those of real-time quantitative polymerase chain reaction(qPCR),real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR), or sequencing. Results: Compared with qPCR or qRT-PCR, the sensitivities of HBV RAA,RSV RT-RAA, and B.pertussis RAA were 97.55%,96.67%, and 100%,respectively,and all of the specificities were 100%.The total coincidence rates were 97.78%(383/392,95%CI:95.63%–98.85%),97.70%(212/217, 95%CI:94.57%–99.16%), and 100%(60/60,95%CI:92.80%–100%),respectively.The Kappa values were 0.977,0.947, and 1,respectively(P<0.05).Regarding the sequencing, the sensitivities of HAdV3 RAA and HAdV7 RAA were 100% and 97.37%, respectively,and all specificities were 100%.The total coincidence rates were 100%(97/97,95%CI:91.58%–100%) and 98.97%(96/97,95%CI:94.39%–99.82%),and the Kappa values were 1 and 0.978 (P<0.05),respectively.Conclusions: With comparable clinical performance, these RAA kits are suitable assays for rapidly detecting pathogens in resource-limited laboratories.

scholarly journals MMP1 is a Promiseful Prognostic Biomarker and Correlating with Immune Infiltrates in Hepatocellular Carcinoma

2021 ◽  
Author(s):  
Lei Dai ◽  
Joseph Muggnyi ◽  
xingchen cai ◽  
shuqi mao ◽  
tongyue zhang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Clinical Performance ◽  
Quantitative Polymerase Chain Reaction ◽  
Human Adenovirus ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Polymerase Chain

Abstract Background: Recombinase-aided amplification(RAA) is a new, simple, and ultrafast isothermal molecular diagnostic technique performed within 30min at 39°C–42°C.In this study, we evaluated the clinical performance of four duplex RAA kits for hepatitis B virus(HBV), human adenovirus 3(HAdV3), human adenovirus 7(HAdV7), and Bordetella pertussis and one duplex reverse-transcription RAA (RT-RAA) kit for respiratory syncytial virus (RSV).Methods: A total of 392 sera and 374 respiratory tract samples were collected from five institutions in four China regions. Each RAA kit’s sensitivity and specificity were compared with those of real-time quantitative polymerase chain reaction(qPCR),real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR), or sequencing. Results: Compared with qPCR or qRT-PCR, the sensitivities of HBV RAA,RSV RT-RAA, and B.pertussis RAA were 97.55%,96.67%, and 100%,respectively,and all of the specificities were 100%.The total coincidence rates were 97.78%(383/392,95%CI:95.63%–98.85%),97.70%(212/217, 95%CI:94.57%–99.16%), and 100%(60/60,95%CI:92.80%–100%),respectively. The Kappa values were 0.977,0.947, and 1,respectively(P<0.05).Regarding the sequencing, the sensitivities of HAdV3 RAA and HAdV7 RAA were 100% and 97.37%, respectively, and all specificities were 100%.The total coincidence rates were 100%(97/97,95%CI:91.58%–100%) and 98.97%(96/97,95%CI:94.39%–99.82%),and the Kappa values were 1 and 0.978 (P<0.05),respectively.Conclusions: With comparable clinical performance, these RAA kits are suitable assays for rapidly detecting pathogens in resource-limited laboratories.

scholarly journals Advanced CRISPR-Cas Effector Enzyme-Based Diagnostics for Infectious Diseases, Including COVID-19

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1356
Author(s):  
Sangha Kwon ◽  
Ha Youn Shin
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Pathogen Detection ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Time Accuracy

Rapid and precise diagnostic tests can prevent the spread of diseases, including worldwide pandemics. Current commonly used diagnostic methods include nucleic-acid-amplification-based detection methods and immunoassays. These techniques, however, have several drawbacks in diagnosis time, accuracy, and cost. Nucleic acid amplification methods are sensitive but time-consuming, whereas immunoassays are more rapid but relatively insensitive. Recently developed CRISPR-based nucleic acid detection methods have been found to compensate for these limitations. In particular, the unique collateral enzymatic activities of Cas12 and Cas13 have dramatically reduced the diagnosis times and costs, while improving diagnostic accuracy and sensitivity. This review provides a comprehensive description of the distinct enzymatic features of Cas12 and Cas13 and their applications in the development of molecular diagnostic platforms for pathogen detection. Moreover, it describes the current utilization of CRISPR-Cas-based diagnostic techniques to identify SARS-CoV-2 infection, as well as recent progress in the development of CRISPR-Cas-based detection strategies for various infectious diseases. These findings provide insights into designing effective molecular diagnostic platforms for potential pandemics.

scholarly journals Detection of SARS-CoV-2 in Saliva by RT-LAMP During a Screening of Workers in Brazil, Including Pre-Symptomatic Carriers

2021 ◽  
Author(s):  
Carlos dos Santos ◽  
Kézia de Oliveira ◽  
Geovana Mendes ◽  
Lívia Silva ◽  
Marcio de Souza Jr. ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Asymptomatic Carrier ◽  
Point Of Care ◽  
Quantitative Polymerase Chain Reaction ◽  
Lamp Assay ◽  
Diagnostic Methods ◽  
Public And Private ◽  
Polymerase Chain ◽  
Increasing Demand

The coronavirus pandemic has been causing damage to many nations, as public and private health systems deteriorate by the increasing demand. Some infected patients have culturable severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) even though not presenting any symptoms, and therefore, are probably able to transmit it. Correctly diagnosing and isolating infected patients is an important step towards preventing new infections. Current diagnostic methods rely mainly on reverse transcription quantitative polymerase chain reaction (RT-qPCR). Methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) have risen as viable alternatives, as they are cheaper and require less infrastructure, they have the potential to be applied in low-resource scenarios and even at point-of-care. Here we report a colorimetric RT‑LAMP assay capable of detecting SARS-CoV-2 in ribonucleic acid (RNA) from saliva. In some cases, the test was able to detect viral RNA before symptom onset and even in a self-reported asymptomatic carrier. It had a limit of detection of 300 copies per reaction and showed a sensitivity of 80%, a specificity of 100%, a general accuracy of 99.59%, and a Cohen’s kappa of 0.887. The possibility of detecting positive cases even before the clinical manifestation shows great potential and can contribute to controlling the pandemic.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

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.

scholarly journals High Frequency of Cryptosporidium hominis Infecting Infants Points to A Potential Anthroponotic Transmission in Maputo, Mozambique

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 293
Author(s):  
Idalécia Cossa-Moiane ◽  
Hermínio Cossa ◽  
Adilson Fernando Loforte Bauhofer ◽  
Jorfélia Chilaúle ◽  
Esperança Lourenço Guimarães ◽  
...  
Keyword(s):  
Public Hospitals ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
P Value ◽  
Cryptosporidium Hominis ◽  
Cryptosporidium Spp ◽  
Pcr Rflp ◽  
Maputo City ◽  
Stool Samples ◽  
Polymerase Chain

Cryptosporidium is one of the most important causes of diarrhea in children less than 2 years of age. In this study, we report the frequency, risk factors and species of Cryptosporidium detected by molecular diagnostic methods in children admitted to two public hospitals in Maputo City, Mozambique. We studied 319 patients under the age of five years who were admitted due to diarrhea between April 2015 and February 2016. Single stool samples were examined for the presence of Cryptosporidium spp. oocysts, microscopically by using a Modified Ziehl–Neelsen (mZN) staining method and by using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) technique using 18S ribosomal RNA gene as a target. Overall, 57.7% (184/319) were males, the median age (Interquartile range, IQR) was 11.0 (7–15) months. Cryptosporidium spp. oocysts were detected in 11.0% (35/319) by microscopy and in 35.4% (68/192) using PCR-RFLP. The most affected age group were children older than two years, [adjusted odds ratio (aOR): 5.861; 95% confidence interval (CI): 1.532–22.417; p-value < 0.05]. Children with illiterate caregivers had higher risk of infection (aOR: 1.688; 95% CI: 1.001–2.845; p-value < 0.05). An anthroponotic species C. hominis was found in 93.0% (27/29) of samples. Our findings demonstrated that cryptosporidiosis in children with diarrhea might be caused by anthroponomic transmission.

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