scholarly journals Application and evaluation of nucleic acid sequence-based amplification, PCR and cryptococcal antigen test for diagnosis of cryptococcosis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yanping Wang ◽  
Mi Yang ◽  
Yun Xia ◽  
Jia Yan ◽  
Jiaqi Zou ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Colloidal Gold ◽  
Detection Method ◽  
Nucleic Acid Sequence ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Control Group ◽  
Pulmonary Cryptococcosis ◽  
K Value ◽  
Cryptococcal Antigen

Abstract Background Cryptococcosis is a major opportunistic invasive mycosis in immunocompromised patients, but it is also increasingly seen in immunocompetent patients. In the early stages of cryptococcosis, limitations of the detection method may hinder the diagnosis. A molecular diagnostic technique based on nucleic acid sequence-based amplification (NASBA) method was developed to fulfil the need for efficient diagnosis of cryptococcosis. Methods We compared the diagnostic performance of NASBA, PCR and cryptococcal antigen (CrAg) test (colloidal gold method) in clinical samples from 25 cryptococcosis patients (including 8 cryptococcal meningoencephalitis and 17 pulmonary cryptococcosis) who were categorized as proven cases (n = 10) and probable cases (n = 15) according to the revised EORTC/MSG definitions. 10 patients with non-Cryptococcus infection and 30 healthy individuals were categorized as control group. Results The lowest detection limit of NASBA was 10 CFU/mL, and RNA of non-target bacteria or fungi was not amplified. The sensitivity of NASBA, PCR and colloidal gold method was 92.00% (95% CI 72.50–98.60%), 64.00% (95% CI 42.62–81.29%), 100.00% (95% CI 83.42–100.00%), and the specificity was 95.00% (95% CI 81.79–99.13%), 80.00% (95% CI 63.86–90.39%) and 82.50% (95% CI 66.64–92.11%) respectively. The highest specificity (97.50%), accuracy (95.38%) and k value (0.90) were achieved when both NASBA and colloidal gold results were positive. Conclusions NASBA is a new alternative detection method for cryptococcosis which is both accurate and rapid without expensive equipment and specialised personnel. It may be used as a tool for confirming current infection as well as monitoring the effectiveness of antifungal treatment. The use of NASBA to detect Cryptococcus RNA in blood samples is of great significance for the diagnosis of pulmonary cryptococcosis. The combination of NASBA and colloidal gold can improve the diagnostic accuracy of cryptococcosis.

scholarly journals Development and Performance verification of colloidal gold labeled SARS-CoV-2 antigen detection method for routine popular screening of COVID-19 with clinical samples in Poland and China

2021 ◽  
Author(s):  
Chuanxiang Guo ◽  
Li Yao ◽  
Fengling Chen ◽  
Chao Zhang ◽  
Wei Chen
Keyword(s):  
Predictive Value ◽  
Colloidal Gold ◽  
Detection Method ◽  
Age Distribution ◽  
Antigen Detection ◽  
Lateral Flow ◽  
Rapid Screening ◽  
Clinical Samples ◽  
Lateral Flow Strip ◽  
And Performance

In this research, we have constructed and optimized the colloidal gold labeled lateral flow strip (LFS) for rapid detection of antigen of SARS-CoV-2 and rapid screening of COVID-19. Based on the constructed and optimized colloidal gold lateral flow strip, the parameters of the LFS have been well evaluated with the clinical samples in the professional labs. The screening performance have also been evaluated from the aspects including the CT values, age distribution and onset of symptoms. Finally, based on the detection results of 420 clinical samples, the LFS can achieve the screening of COVID-19 with the positive percentage agreement (PPA, sensitivity), negative percent agreement (NPA, specificity), the positive predictive value (PPV) and the negative predictive value (NPV) of 96.8%, 100%, 100% and 96.6%, respectively, indicating the powerful potential for practical screening applications in pandemic control. Of great significance, this developed SARS-CoV-2 antigen detection method has also been successfully utilized for screening of delta-variant of SARS-CoV-2.

Sensitivity of detection of rhinoviruses in spiked clinical samples by nucleic acid sequence-based amplification in the presence of an internal control

2006 ◽  
Vol 66 (1) ◽  
pp. 73-78 ◽  
Author(s):  
Katherine Loens ◽  
Margareta Ieven ◽  
Stefaan Pattyn ◽  
Peter Sillekens ◽  
Herman Goossens
Keyword(s):  
Nucleic Acid ◽  
Internal Control ◽  
Nucleic Acid Sequence ◽  
Clinical Samples

scholarly journals Evaluation of real-time nucleic acid sequence-based amplification for detection of Chikungunya virus in clinical samples

2009 ◽  
Vol 58 (9) ◽  
pp. 1168-1172 ◽  
Author(s):  
J.-N. Telles ◽  
K. Le Roux ◽  
P. Grivard ◽  
G. Vernet ◽  
A. Michault
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Chikungunya Virus ◽  
False Negative ◽  
Nucleic Acid Sequence ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Plasma Samples ◽  
Rt Pcr ◽  
Nucleic Acid Extraction

The Chikungunya virus (CHIKV) is a member of the genus Alphavirus that is transmitted to humans by Aedes mosquitoes. In 2005 and 2006, the Indian Ocean island of La Réunion was hit with an unprecedented CHIKV fever outbreak that infected 300 000 people. In the present study, we describe the evaluation of real-time nucleic acid sequence-based amplification (RT-NASBA) for the detection of CHIKV in clinical samples. A co-extracted and co-amplified chimerical CHIKV RNA sequence was used as an internal control to eliminate false-negative results. The detection threshold of the assay was determined from quantified CHIKV-positive plasma, and estimated to be 200 copies per NASBA reaction. The specificity of the assay was determined using blast analyses and non-cross-reactivity using an O'nyong-nyong virus culture and 250 CHIKV RT-PCR-negative plasma samples. A 100 % specificity was found and no invalid result was obtained, showing the good quality of the nucleic acid extraction. The assay was then evaluated using 252 CHIKV-positive RT-PCR plasma samples. The samples were all tested positive, including those with low viral load. This evaluation showed that the RT-NASBA is a rapid (5 h from sample nucleic acid extraction to detection), sensitive, specific and reliable method for the routine diagnosis of CHIKV in clinical samples.

scholarly journals Nucleic Acid Sequence-Based Amplification with Oligochromatography for Detection of Trypanosoma brucei in Clinical Samples

2008 ◽  
Vol 47 (3) ◽  
pp. 630-635 ◽  
Author(s):  
C. M. Mugasa ◽  
T. Laurent ◽  
G. J. Schoone ◽  
P. A. Kager ◽  
G. W. Lubega ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Trypanosoma Brucei ◽  
Nucleic Acid Sequence ◽  
Clinical Samples

scholarly journals Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR

2001 ◽  
Vol 67 (12) ◽  
pp. 5593-5600 ◽  
Author(s):  
Julie Jean ◽  
Burton Blais ◽  
André Darveau ◽  
Ismaı̈l Fliss
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Nucleic Acid Sequence ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Dot Blot ◽  
Reverse Transcription Pcr ◽  
Dot Blot Assay

ABSTRACT A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 102 PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 104PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

scholarly journals Detection of Mycoplasma pneumoniae in Spiked Clinical Samples by Nucleic Acid Sequence-Based Amplification

2002 ◽  
Vol 40 (4) ◽  
pp. 1339-1345 ◽  
Author(s):  
K. Loens ◽  
D. Ursi ◽  
M. Ieven ◽  
P. van Aarle ◽  
P. Sillekens ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Mycoplasma Pneumoniae ◽  
Nucleic Acid Sequence ◽  
Clinical Samples

Molecular Diagnostics

2019 ◽  
Author(s):  
Duncan Clark ◽  
Mark Wilks
Keyword(s):  
Nucleic Acid ◽  
Molecular Diagnostics ◽  
Diagnostic Tests ◽  
Clinical Sample ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Molecular Test ◽  
Block Based ◽  
Set Up

Molecular diagnostics in infection generally relate to the detection and/ or characterization of nucleic acid sequences of infectious agents in clinical samples which are used to provide: ● A laboratory diagnosis. ● A means of monitoring patients at risk of developing disease caused by a particular infection. ● A method to predict through genotypic analysis the susceptibility or resistance to appropriate treatments. ● A measurement of the response to therapy. A few key laboratory techniques underpin the majority of molecular diagnostic tests that are currently used in the field of infection, and include: ● Block-based polymerase chain reaction (PCR). ● Real-time PCR, including quantification. ● Strand displacement amplification. ● Transcription mediated amplification. ● DNA sequencing. These can be commercially sourced, which has the advantage of CE marking, or developed in-house, sometimes referred to as laboratory developed tests (LDTs). Whatever the source, the underlying principles are often the same and rigorous evaluation and validation is required for the adoption of any molecular test in the diagnostic laboratory. The majority of molecular diagnostic tests require the amplification of a specific DNA sequence and its subsequent detection by a variety of means. As such, small sequences of DNA from the infectious agent are amplified from a relatively low copy number in the clinical sample. For example, after thirty to forty cycles of PCR, a single copy of a sequence can theoretically be amplified to over a billion copies. This PCR product, commonly termed amplicon, can provide a template for any further testing with the same PCR test and therefore potentially act as a source for false positive results. Molecular diagnostic laboratories have requirements to keep the different stages of the molecular test separate and minimize the risk of amplicon contamination. Most facilities will have a ‘clean PCR laboratory’ that is used to store the clean reagents such as primers, probes, enzyme mastermixes, and no clinical samples, nucleic extracts, or amplification reactions are ever taken into this environment. Another laboratory is used for the nucleic acid extraction of the clinical samples and this environment is often used to set up the PCR reactions.

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.

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