Nucleic acid amplification-based methods for microRNA detection

2015 ◽  
Vol 7 (6) ◽  
pp. 2258-2263 ◽  
Author(s):  
Hui-Ling Chen ◽  
Meng-Meng Guo ◽  
Hao Tang ◽  
Zhan Wu ◽  
Li-Juan Tang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Basic Principles ◽  
Microrna Detection

This review traces the basic principles of several nucleic acid amplification-based microRNA detection methods that have been developed in recent three years.

Development of a Molecular Method for the Detection of Enteric Viruses in Oysters

1995 ◽  
Vol 58 (12) ◽  
pp. 1357-1362 ◽  
Author(s):  
LEE-ANN JAYKUS ◽  
RICARDO DE LEON ◽  
MARK D. SOBSEY
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Hepatitis A Virus ◽  
Enteric Virus ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Ammonium Bromide ◽  
Rt Pcr ◽  
Initial Inoculum ◽  
Ctab Precipitation

Detection of enteric virus contamination of shellfish is limited by current methodology, which is time-consuming, tedious, and lacking in sensitivity due to reliance on cell culture infectivity. Alternative detection methods based on nucleic acid amplification have been hampered by high sample volumes and the presence of enzymatic inhibitors. The goal of this study was to develop methods to purify and concentrate intact virions from oyster extracts to a volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase-polymerase chain reaction (RT-PCR). Fifty-gram oyster samples were homogenized and processed by standard adsorption-elution precipitation methodology and then seeded with 105 PFU of poliovirus 1 (PV1) or hepatitis A virus (HAV). Seeded viruses were concentrated by fluorocarbon extraction, polyethylene glycol (PEG) precipitation, chloroform extraction, and cetyltrimethyl ammonium bromide (CTAB) precipitation to a volume of 100 μl with removal of RT-PCR inhibitors. Virus recovery after elution of PEG precipitates was 50% for PVI and IS to 20% for HAV as evaluted by cell culture infectivity. The CTAB precipitation step yielded a concentrated sample which was directly compatible with RT-PCR reactions and capable of detecting about 100 placque=forming units (PFU) of PVl or HAV. When 50-g oyster extracts were seeded and processed by the entire concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 104 PFU of HAV and 103 PFU of PV1, with recoveries of 1 to 5% of seeded viruses.

scholarly journals Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

2020 ◽  
Author(s):  
Soon Keong Wee ◽  
Suppiah Paramalingam Sivalingam ◽  
Eric Peng Huat Yap
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Cost Effective ◽  
Turnaround Time ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
N Gene ◽  
Direct Pcr ◽  
Viral Lysis

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as 6 RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification and detection are achieved in a single-tube homogeneous reaction within 36 minutes. This minimized hands-on time, reduces turnaround-time for sample-to-result and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening and research in countries and regions where laboratory capabilities are limiting.

scholarly journals Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 664 ◽  
Author(s):  
Soon Keong Wee ◽  
Suppiah Paramalingam Sivalingam ◽  
Eric Peng Huat Yap
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Cost Effective ◽  
Turnaround Time ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
N Gene ◽  
Direct Pcr ◽  
Viral Lysis

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting.

9. DEVELOPMENT AND VALIDATION OF A NOVEL GEL-BASED URINE TRANSPORT SYSTEM FOR USE IN CHLAMYDIA TRACHOMATIS PCR BASED DIAGNOSIS

Sexual Health ◽  
2007 ◽  
Vol 4 (4) ◽  
pp. 287
Author(s):  
S. Bialasiewicz
Keyword(s):  
Nucleic Acid ◽  
Chlamydia Trachomatis ◽  
Ease Of Use ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Diagnostic Laboratory ◽  
Initial Development ◽  
Home Setting ◽  
Development And Validation ◽  
Urine Testing

Background: Chlamydia trachomatis infection rates have increased within Australia over the past several years, including persistently high incidences in known risk groups. The development of novel C. trachomatis detection methods which can be self-collected and mailed in a plain envelope presents significant opportunities for increasing access to urine testing across Australia, particularly those who are geographically or socially isolated and have limited or impeded access to mainstream health services. Aim: The purpose of the study was to develop a urine transportation system which retains comparable sensitivity to standard sampling methods, is easy and safe to use by the average person within a home setting, and which complies with regulations concerning the transport of biological specimen through regular mail. Results/Discussion: An expanding-matrix based method was developed in which a small amount of urine is applied to a dry mixture of a super absorbent polymer and nucleic acid stabiliser, yielding a dry gel. The gel can then be subsequently treated in the diagnostic laboratory to release the reconstituted urine, from which nucleic acid can be extracted using standard methods. Once extracted, the sample can be utilised in a nucleic acid amplification based C. trachomatis diagnostic assay. The clinical sensitivity of the gel-matrix was found to be comparable to that of standard urine transport methods. The applicability of the gel for use by the public in a home collection setting was deemed appropriate due to the non-toxic nature of the matrix materials, ease of use, and the basic packing and postage requirements. The dry gel form of the urine and packaging complied with Australia Post standard postage requirements. Results of the initial development and validation of the gel matrix will be presented.

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 Advances in nucleic acid-based detection methods.

1992 ◽  
Vol 5 (4) ◽  
pp. 370-386 ◽  
Author(s):  
M J Wolcott
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Background Information ◽  
Clinical Settings ◽  
Chain Reaction ◽  
Detection Techniques ◽  
Amplification Technique ◽  
Polymerase Chain

Laboratory techniques based on nucleic acid methods have increased in popularity over the last decade with clinical microbiologists and other laboratory scientists who are concerned with the diagnosis of infectious agents. This increase in popularity is a result primarily of advances made in nucleic acid amplification and detection techniques. Polymerase chain reaction, the original nucleic acid amplification technique, changed the way many people viewed and used nucleic acid techniques in clinical settings. After the potential of polymerase chain reaction became apparent, other methods of nucleic acid amplification and detection were developed. These alternative nucleic acid amplification methods may become serious contenders for application to routine laboratory analyses. This review presents some background information on nucleic acid analyses that might be used in clinical and anatomical laboratories and describes some recent advances in the amplification and detection of nucleic acids.

scholarly journals Laboratory Diagnostic Tools for Syphilis: Current Status and Future Prospects

2021 ◽  
Vol 10 ◽  
Author(s):  
Yuting Luo ◽  
Yafeng Xie ◽  
Yongjian Xiao
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Detection Methods ◽  
Current Status ◽  
Morphological Observation ◽  
Diagnostic Tools ◽  
Clinical Samples ◽  
Serologic Tests ◽  
Number Of Patients ◽  
Infectivity Test

With the increasing number of patients infected with syphilis in the past 20 years, early diagnosis and early treatment are essential to decline syphilis prevalence. Owing to its diverse manifestations, which may occur in other infections, the disease often makes clinicians confused. Therefore, a sensitive method for detecting T. pallidum is fundamental for the prompt diagnosis of syphilis. Morphological observation, immunohistochemical assay, rabbit infectivity test, serologic tests, and nucleic acid amplification assays have been applied to the diagnosis of syphilis. Morphological observation, including dark-field microscopy, silver-staining, and direct fluorescent antibody staining for T. pallidum, can be used as a direct detection method for chancre specimens in primary syphilis. Immunohistochemistry is a highly sensitive and specific assay, especially in the lesion biopsies from secondary syphilis. Rabbit infectivity test is considered as a sensitive and reliable method for detecting T. pallidum in clinical samples and used as a historical standard for the diagnosis of syphilis. Serologic tests for syphilis are widely adopted using non-treponemal or treponemal tests by either the traditional or reverse algorithm and remain the gold standard in the diagnosis of syphilis patients. In addition, nucleic acid amplification assay is capable of detecting T. pallidum DNA in the samples from patients with syphilis. Notably, PCR is probably a promising method but remains to be further improved. All of the methods mentioned above play important roles in various stages of syphilis. This review aims to provide a summary of the performance characteristics of detection methods for syphilis.

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