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 SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases

2020 ◽  
Author(s):  
Mujahed I. Mustafa ◽  
Abdelrafie M. Makhawi
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Pathogen Detection ◽  
Emerging Infectious Diseases ◽  
Cost Effective ◽  
Detection Methods ◽  
Diagnostic Tools ◽  
Nucleic Acid Detection ◽  
Nucleic Acid Binding

Infectious diseases are one of the most intimidating threats to human race, responsible for an immense burden of disabilities and deaths. Rapid diagnosis and treatment of infectious diseases is a better understanding of its pathogenesis. According to WHO, the ideal approach for detecting foreign pathogens should be rapid, specific, sensitive, instrument-free and cost-effective. Nucleic acid pathogen detection methods, typically PCR have numerous limitations, such as highly sophisticated equipments, reagents, and trained personnel rely on well-established laboratories beside time-consuming. Thus, there is a crucial need to develop novel nucleic acid detection tools with rapid, specific, sensitive, and cost-effective, particularly ones that can be used for versatile point-of-care diagnostic applications. Two new methods exploit on unpredicted in vitro properties CRISPR-Cas effectors, turning activated nucleases into basic amplifiers of a specific nucleic-acid binding event. These effectors are attached with a diversity of reporters and utilized in tandem with present of isothermal amplification approaches to create sensitive identification in multiple field deployable formats. Although still in their beginning, yet SHERLOCK and DETECTR technologies are potential methods for rapid detection and identification of infectious disease, with ultra-sensitive tests that don't require a lot of complicated processing. This review described SHERLOCK and DETECTR technologies beside their properties, functions, and perspectives to become the ultimate diagnostic tools for diagnosing infectious diseases and curbing disease outbreaks.

scholarly journals IsoPCR: An Analytically Sensitive, Nested, Multiplex Nucleic Acid Amplification Method

2013 ◽  
Vol 59 (2) ◽  
pp. 436-439 ◽  
Author(s):  
Martin Jensen Søe ◽  
Mikkel Rohde ◽  
Jens Mikkelsen ◽  
Peter Warthoe
Keyword(s):  
Nucleic Acid ◽  
Candida Glabrata ◽  
Simultaneous Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Amplification Method

BACKGROUND Nucleic acid tests that can simultaneously detect multiple targets with high sensitivity, specificity, and speed are highly desirable. To meet this need, we developed a new approach we call the isoPCR method. METHODS The isoPCR method is a 2-stage nested-like nucleic acid amplification method that combines a single multiplex preamplification PCR with subsequent distinct detection of specific targets by use of isothermal amplification. We compared isoPCR to nested quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and nested LAMP (PCR followed by LAMP), for detection of DNA from Candida glabrata. We evaluated the method's multiplex capability for detecting low copy numbers of pathogens commonly involved in sepsis. RESULTS IsoPCR provided detection of 1 copy of Candida glabrata, an LOD that was 5-fold lower than a nested qPCR assay (5 copies), while the amplification time was simultaneously halved. Similarly, the LOD for isoPCR was lower than that for a LAMP assay (1000 copies) and a nested LAMP assay (5 copies). IsoPCR required recognition of 6 regions for detection, thereby providing a theoretically higher specificity compared to nested qPCR (4 regions). The isoPCR multiplexing capability was demonstrated by simultaneous detection of 4 pathogens with individual LODs of 10 copies or fewer. Furthermore, the specificity of isoPCR was demonstrated by successful pathogen detection from samples with more than 1 pathogen present. CONCLUSIONS IsoPCR provides a molecular diagnostic tool for multiplex nucleic acid detection, with an LOD down to 1 copy, high theoretical specificity, and halving of the amplification time compared to a nested qPCR assay.

Relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory.

1997 ◽  
Vol 10 (2) ◽  
pp. 242-256 ◽  
Author(s):  
M Ieven ◽  
H Goossens
Keyword(s):  
Nucleic Acid ◽  
Respiratory Tract ◽  
Respiratory Tract Infections ◽  
Clinical Laboratory ◽  
Pneumocystis Carinii ◽  
Diagnostic Techniques ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Amplification Techniques ◽  
Tract Infections

Clinical laboratories are increasingly receiving requests to perform nucleic acid amplification tests for the detection of a wide variety of infectious agents. In this paper, the efficiency of nucleic acid amplification techniques for the diagnosis of respiratory tract infections is reviewed. In general, these techniques should be applied only for the detection of microorganisms for which available diagnostic techniques are markedly insensitive or nonexistent or when turnaround times for existing tests (e.g., viral culture) are much longer than those expected with amplification. This is the case for rhinoviruses, coronaviruses, and hantaviruses causing a pulmonary syndrome, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Coxiella burnetii. For Legionella spp. and fungi, contamination originating from the environment is a limiting factor in interpretation of results, as is the difficulty in differentiating colonization and infection. Detection of these agents in urine or blood by amplification techniques remains to be evaluated. In the clinical setting, there is no need for molecular diagnostic tests for the diagnosis of Pneumocystis carinii. At present, amplification methods for Mycobacterium tuberculosis cannot replace the classical diagnostic techniques, due to their lack of sensitivity and the absence of specific internal controls for the detection of inhibitors of the reaction. Also, the results of interlaboratory comparisons are unsatisfactory. Furthermore, isolates are needed for susceptibility studies. Additional work remains to be done on sample preparation methods, comparison between different amplification methods, and analysis of results. The techniques can be useful for the rapid identification of M. tuberculosis in particular circumstances, as well as the rapid detection of most rifampin-resistant isolates. The introduction of diagnostic amplification techniques into a clinical laboratory implies a level of proficiency for excluding false-positive and false-negative results.

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 Infectious Diseases Society of America Guidelines on the Diagnosis of Coronavirus Disease 2019

2020 ◽  
Author(s):  
Kimberly E Hanson ◽  
Angela M Caliendo ◽  
Cesar A Arias ◽  
Janet A Englund ◽  
Mark J Lee ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Universal Access ◽  
Nucleic Acid Amplification ◽  
Evaluation Methodology ◽  
Molecular Diagnostic ◽  
Evidence Based ◽  
Care Hospital ◽  
Nucleic Acid Testing ◽  
Asymptomatic Individuals

Abstract IDSA Disclaimer As of the time of this publication, updates have been made to IDSA’s Guidelines on the Diagnosis of COVID-19. For the most updated version of these guidelines, please go to http://www.idsociety.org/covid19guidelines. Background Accurate molecular diagnostic tests are necessary for confirming a diagnosis of coronavirus disease 2019 (COVID-19). Direct detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acids in respiratory tract specimens informs patient, healthcare institution, and public health–level decision-making. The number of available SARS-CoV-2 nucleic acid detection tests is rapidly increasing, as is the COVID-19 diagnostic literature. Thus, the Infectious Diseases Society of America (IDSA) recognized a significant need for frequently updated systematic reviews of the literature to inform evidence-based best practice guidance. Objective The IDSA’s goal was to develop an evidence-based diagnostic guidelines to assist clinicians, clinical laboratorians, patients, and policy makers in decisions related to the optimal use of SARS-CoV-2 nucleic acid amplification tests. In addition, the society provides a conceptual framework for understanding molecular diagnostic test performance, discusses the nuance of test result interpretation in a variety of practice settings, and highlights important unmet research needs in the COVID-19 diagnostic testing arena. Methods IDSA convened a multidisciplinary panel of infectious diseases clinicians, clinical microbiologists, and experts in systematic literature review to identify and prioritize clinical questions and outcomes related to the use of SARS-CoV-2 molecular diagnostics. Grading of Recommendations Assessment, Development and Evaluation methodology was used to assess the certainty of evidence and make testing recommendations. Results The panel agreed on 15 diagnostic recommendations. Conclusions Universal access to accurate SARS-CoV-2 nucleic acid testing is critical for patient care, hospital infection prevention, and the public response to the COVID-19 pandemic. Information on the clinical performance of available tests is rapidly emerging, but the quality of evidence of the current literature is considered low to very low. Recognizing these limitations, the IDSA panel weighed available diagnostic evidence and recommends nucleic acid testing for all symptomatic individuals suspected of having COVID-19. In addition, testing is recommended for asymptomatic individuals who have had a known or suspected contact with a COVID-19 case. Testing asymptomatic individuals without known exposure is suggested when the results will impact isolation/quarantine/personal protective equipment usage decisions, dictate eligibility for surgery, or inform administration of immunosuppressive therapy. Ultimately, prioritization of testing will depend on institution-specific resources and the needs of different patient populations.

scholarly journals Different Laboratory Diagnosis methods of COVID-19: A Systematic Review

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Navid Omidifar ◽  
Kamran Bagheri Lankarani ◽  
Mohsen Moghadami ◽  
Mansoureh Shokripour ◽  
Mostafa Chashmpoosh ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Laboratory Diagnosis ◽  
Diagnostic Techniques ◽  
Disease Spread ◽  
Detection Methods ◽  
Nucleic Acid Detection ◽  
Laboratory Equipment ◽  
Reactive Protein ◽  
Pcr Method ◽  
Conferences And Congresses

: The virus causing COVID-19 disease is known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease spread rapidly and was transmitted like a contagious disease throughout China, and then it gradually spread in other parts of the world. Accordingly, the rapid and accurate detection of the SARS-CoV-2 virus plays an essential role in selecting timely treatments, saving lives, and preventing the spread of the disease. This study summarizes the methods used to identify coronavirus nucleic acid. The effectiveness of coronavirus nucleic acid detection kits by different samples and the performance of other diagnostic techniques are also addressed in this study. We searched Embase, Google Scholar, MEDLINE, Web of Science, Scopus, and PubMed databases as well as the references of all relevant articles in English published during 2019 - 2020 using keywords related to COVID-19, detection kits, and respiratory failure and proceedings from relevant conferences and congresses. The authors collected the relevant reports, and each of the authors independently reviewed the data published in different studies. The results of previous studies indicated that the diagnosis methods of the COVID-19 disease are the RT-PCR method, ELISA kits, quick tests, white blood cell count, C-reactive protein (CRP) levels, other laboratory factors and antigenic detection methods. Given the sensitivity and specificity of these methods at different periods using different samples, the disease interpretation can be performed accurately. The findings showed that proper laboratory equipment and appropriate laboratory kits are necessary for the rapid and precise identification of COVID-19.

scholarly journals Molecular diagnostics of infectious diseases

1997 ◽  
Vol 43 (11) ◽  
pp. 2021-2038 ◽  
Author(s):  
Yi-Wei Tang ◽  
Gary W Procop ◽  
David H Persing
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Direct Detection ◽  
Clinical Chemistry ◽  
Gene Mutations ◽  
Diagnostic Techniques ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Molecular Diagnostic Techniques ◽  
Plasmid Profiling

Abstract Over the past several years, the development and application of molecular diagnostic techniques has initiated a revolution in the diagnosis and monitoring of infectious diseases. Microbial phenotypic characteristics, such as protein, bacteriophage, and chromatographic profiles, as well as biotyping and susceptibility testing, are used in most routine laboratories for identification and differentiation. Nucleic acid techniques, such as plasmid profiling, various methods for generating restriction fragment length polymorphisms, and the polymerase chain reaction (PCR), are making increasing inroads into clinical laboratories. PCR-based systems to detect the etiologic agents of disease directly from clinical samples, without the need for culture, have been useful in rapid detection of unculturable or fastidious microorganisms. Additionally, sequence analysis of amplified microbial DNA allows for identification and better characterization of the pathogen. Subspecies variation, identified by various techniques, has been shown to be important in the prognosis of certain diseases. Other important advances include the determination of viral load and the direct detection of genes or gene mutations responsible for drug resistance. Increased use of automation and user-friendly software makes these technologies more widely available. In all, the detection of infectious agents at the nucleic acid level represents a true synthesis of clinical chemistry and clinical microbiology techniques.

scholarly journals KOMPARASI ANTARA POLYMERASE CHAIN REACTION (PCR) DAN LOOPMEDIATED ISOTHERMAL AMPLIFICATION (LAMP) DALAM DIAGNOSIS MOLEKULER

2016 ◽  
Vol 3 (2) ◽  
pp. 145
Author(s):  
Anggun Feranisa
Keyword(s):  
Infectious Disease ◽  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Infectious Diseases ◽  
Diagnostic Method ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
Amplification Method ◽  
Polymerase Chain

Background: Molecular diagnostic is an emerging diagnostic method inpersonalized medicine/dentistry era. Usually, it uses nucleic acid amplificationmethod to detect various diseases. PCR is conventional nucleic acid amplification method. However, due to an urgency in infectious diseases’ diagnotic method, scientists developed LAMP as new nucleic acid amplification method.Discussion: There are various experiments used to develop LAMP as infectious diseases diagnostic method compared to PCR. The results are LAMP more sensitive, specific, rapid, and inexpensive than PCR.Conclusion: Both PCR and LAMP can be used as molecular diagnostic tools.LAMP prefer to used as infectious disease diagnostic method in poor anddeveloping countries.

Advances in Molecular Technologies and Platforms for the Diagnosis of Infectious Diseases

2013 ◽  
Vol 810 ◽  
pp. 77-125
Author(s):  
M. Rubayet Hasan
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Potential Method ◽  
Diagnostic Methods ◽  
Control Measures ◽  
Nucleic Acid Amplification ◽  
Microbial Pathogens ◽  
Immunological Methods ◽  
Clinical Samples ◽  
Single Test

nfectious microbial pathogens constitute the largest cause of morbidity and mortality worldwide. Early diagnosis and rapid infection control measures can lead to improved outcomes, earlier discharges and reduced nosocomial infections. Conventional diagnostic methods for infectious diseases such as microscopy, culture, and immunological methods, in most cases, are not universally applicable, less sensitive and could take from days to months to complete depending on the pathogen. Molecular assays based on nucleic acids such as polymerase chain reaction (PCR) have improved the sensitivity, specificity and turn-around time in diagnostic microbiology laboratories. These tests are particularly important to detect very low levels of pathogens in clinical samples, and for organisms that have long half-lives or are non-culturable. However, individual molecular tests are available for only a limited number of the more common infectious agents. Moreover, infectious disease events arising from novel pathogens or genetic variants have significantly increased, recently, for which, routine diagnostic methods are not yet available. Therefore, molecular methods and technologies capable of detecting multiple pathogens in a single test have become available over the last few years. Although, these methods are based on the conventional nucleic acid amplification and hybridization chemistry, enhanced multiplexing capability has been achieved through innovations in nucleic acid labeling techniques, and post-amplification analytic methods and instrumentation. The availability of these test kits brought a new level of convenience to the physicians ordering practices, and to the laboratory personnel, as they require very little hands on time. However, these tests are yet unaffordable to many laboratories, and in many cases, the sensitivity is poor compared to that of single-target, real-time PCR assays. Looking into the future, the revolutionary, next generation sequencing (NGS) technology is now being considered as a potential method for rapid identification of hundreds of pathogens, in an unbiased manner, with a single test that could significantly benefit patients who are critically ill with undiagnosed disease.

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.

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