Development of a high-sensitivity nested PCR assay for the detection of Clostridium piliforme in clinical samples

2010 ◽  
Vol 185 (2) ◽  
pp. 222-224 ◽  
Author(s):  
Alisson Niepceron ◽  
Dominique Licois
Keyword(s):  
Nested Pcr ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Clostridium Piliforme

scholarly journals Evaluation of Different PCR Assay Formats for Sensitive and Specific Detection of SARS-CoV-2 RNA

2020 ◽  
Author(s):  
Jeremy Ratcliff ◽  
Dung Nguyen ◽  
Monique Andersson ◽  
Peter Simmonds
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Nested Pcr ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Copy Detection ◽  
Accurate Identification ◽  
Laboratory Equipment ◽  
Rna Transcripts

ABSTRACTAccurate identification of individuals infected with SARS-CoV-2 is crucial for efforts to control the ongoing COVID-19 pandemic. Polymerase chain reaction (PCR)-based assays are the gold standard for detecting viral RNA in patient samples and are used extensively in clinical settings. Most currently used quantitative PCR (RT-qPCRs) rely upon real-time detection of PCR product using specialized laboratory equipment. To enable the application of PCR in resource-poor or non-specialist laboratories, we have developed and evaluated a nested PCR method for SARS-CoV-2 RNA using simple agarose gel electrophoresis for product detection. Using clinical samples tested by conventional qPCR methods and RNA transcripts of defined RNA copy number, the nested PCR based on the RdRP gene demonstrated high sensitivity and specificity for SARS-CoV-2 RNA detection in clinical samples, but showed variable and transcript length-dependent sensitivity for RNA transcripts. Samples and transcripts were further evaluated in an additional N protein real-time quantitative PCR assay. As determined by 50% endpoint detection, the sensitivities of three RT-qPCRs and nested PCR methods varied substantially depending on the transcript target with no method approaching single copy detection. Overall, these findings highlight the need for assay validation and optimization and demonstrate the inability to precisely compare viral quantification from different PCR methodologies without calibration.

scholarly journals Detection of Aspergillus DNA by a nested PCR assay is able to improve the diagnosis of invasive aspergillosis in paediatric patients

2009 ◽  
Vol 58 (10) ◽  
pp. 1291-1297 ◽  
Author(s):  
Margit Hummel ◽  
Birgit Spiess ◽  
Julia Roder ◽  
Gregor von Komorowski ◽  
Matthias Dürken ◽  
...  
Keyword(s):  
Fungal Infection ◽  
Invasive Aspergillosis ◽  
Nested Pcr ◽  
Fungal Infections ◽  
High Sensitivity ◽  
Clinical Findings ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Imaging Results ◽  
Adolescent Patients

Fungal infections are a leading cause of morbidity and mortality in severely immunocompromised patients and have been increasing in incidence in recent years. Invasive aspergillosis (IA) is the most common filamentous fungal infection and is, in adults as well as in children, difficult to diagnose. Several PCR assays to detect Aspergillus DNA have been established, but so far, studies on molecular tools for the diagnosis of IA in children are few. We evaluated the results of a nested PCR assay to detect Aspergillus DNA in clinical samples from paediatric and adolescent patients with suspected IA. Blood and non-blood samples from immunocompromised paediatric and adolescent patients with suspected invasive fungal infection were sent for processing Aspergillus PCR to our laboratory. PCR results from consecutive patients from three university children's hospitals investigated between November 2000 and January 2007 were evaluated. Fungal infections were classified according to the EORTC classification on the grounds of clinical findings, microbiology and radio-imaging results. Two hundred and ninety-one samples from 71 patients were investigated for the presence of Aspergillus DNA by our previously described nested PCR assay. Two, 3 and 34 patients had proven, probable and possible IA, respectively. Sensitivity (calculated from proven and probable patients, n=5) and specificity (calculated from patients without IA, n=32) rates of the PCR assay were 80 and 81 %, respectively. Our nested PCR assay was able to detect Aspergillus DNA in blood, cerebrospinal fluid and bronchoalveolar lavage samples from paediatric and adolescent patients with IA with high sensitivity and specificity rates.

scholarly journals Validation and Clinical Application of a Molecular Method for Identification of Histoplasma capsulatum in Human Specimens in Colombia, South America

2009 ◽  
Vol 17 (1) ◽  
pp. 62-67 ◽  
Author(s):  
Cesar Muñoz ◽  
Beatriz L. Gómez ◽  
Angela Tobón ◽  
Karen Arango ◽  
Angela Restrepo ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Predictive Value ◽  
Histoplasma Capsulatum ◽  
Clinical Samples ◽  
Healthy Individuals ◽  
Molecular Method ◽  
Pcr Assay ◽  
Molecular Assays ◽  
Panfungal Pcr ◽  
Negative Controls

ABSTRACT The conventional means of diagnosis of histoplasmosis presents difficulties because of the delay to the time that the diagnosis is made, indicating the need for the implementation of molecular assays. We evaluated 146 clinical samples from 135 patients suspected of having histoplasmosis using a previously reported nested PCR assay for the H istoplasma capsulatum-specific 100-kDa protein (the Hc100 PCR). In order to determine the specificity of this molecular test, we also used samples from healthy individuals (n = 20), patients suspected of having respiratory disease with negative fungal cultures (n = 29), and patients with other proven infections (n = 60). Additionally, a sizable collection of DNA from cultures of H. capsulatum and other medically relevant pathogens was studied. A panfungal PCR assay that amplified the internal transcribed spacer 2 region was also used to identify all fungal DNAs. All PCR-amplified products were sequenced. Of the 146 clinical samples, 67 (45.9%) were positive by culture and PCR, while 9 samples negative by culture were positive by PCR. All the sequences corresponding to the 76 amplified products presented ≥98% identity with H. capsulatum. The Hc100 PCR exhibited a sensitivity of 100% and specificities of 92.4% and 95.2% when the results were compared to those for the negative controls and samples from other proven clinical entities, respectively; the positive predictive value was 83% and the negative predictive value was 100%; the positive and negative likelihood rates were 25 and 0, respectively. These results suggest that the Hc100 nested PCR assay for the detection of H. capsulatum DNA is a useful test in areas where mycosis caused by this organism is endemic.

scholarly journals Toward point-of-care diagnostics of Candida auris

2020 ◽  
Author(s):  
Geoffrey Mulberry ◽  
Sudha Chaturvedi ◽  
Vishnu Chaturvedi ◽  
Brian N. Kim
Keyword(s):  
New York ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Impact ◽  
Point Of Care ◽  
Low Cost ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Candida Auris

AbstractCandida auris is a multidrug-resistant yeast that presents global health threat for the hospitalized patients. Early diagnostic of C. auris is crucial in control, prevention, and treatment. Candida auris is difficult to identify with standard laboratory methods and often can be misidentified leading to inappropriate management. A newly-devised real-time PCR assay played an important role in the ongoing investigation of the C. auris outbreak in New York metropolitan area. The assay can rapidly detect C. auris DNA in surveillance and clinical samples with high sensitivity and specificity, and also useful for confirmation of C. auris cultures. Despite its positive impact, the real-time PCR assay is difficult to deploy at frontline laboratories due to high-complexity set-up and operation. Using a low-cost handheld real-time PCR device, we show that the C. auris can potentially be identified in a low-complexity assay without the need for high-cost equipment. An implementation of low-cost real-time PCR device in hospitals and healthcare facilities is likely to accelerate the diagnosis of C. auris and for control of the global epidemic.

Whole-genome sequence-guided PCR for the rapid identification of thePseudomonas aeruginosa ST175 high-risk clone directly from clinical samples

2020 ◽  
Author(s):  
Gabriel Cabot ◽  
Paula Lara-Esbrí ◽  
Xavier Mulet ◽  
Antonio Oliver
Keyword(s):  
High Risk ◽  
Sensitivity And Specificity ◽  
Genome Sequence ◽  
Treatment Strategies ◽  
High Sensitivity ◽  
Whole Genome Sequence ◽  
Clinical Samples ◽  
Whole Genome ◽  
Pcr Assay ◽  
Pcr Targeting

Abstract Objectives Pseudomonas aeruginosa frequently show MDR/XDR profiles, which are associated with worldwide-disseminated high-risk clones (HRCs). We developed a PCR assay for the detection in clinical samples of ST175, an HRC that is widespread in European countries. Methods The whole-genome sequence was obtained for one ST175 isolate using a PacBio RSII sequencer. Reads from multiple isolates belonging to ST175 and the PAO1 reference strain were mapped against the ST175 genome to identify potentially specific regions. Once curated, using the BLAST database to search for the presence of those regions in any other organism, we designed a specific PCR for the detection of ST175. Results Assembly of the ST175 PacBio-sequenced genome resulted in three contigs with a total length of 7 087 985 bases, encoding 6566 coding sequences. Specific regions for ST175 genomes were detected and a PCR targeting a 318 bp fragment located within a 3177 bp ORF coding for a putative reverse transcriptase was designed. The PCR test was first evaluatedin silico against 229 XDRP. aeruginosa genomes (73 ST175) from two multicentre studies, yielding 100% sensitivity and specificity. Then, the PCR was evaluatedin vitro in 25 isolates (12 ST175) and in 120 clinical samples (30 urine samples, 30 blood cultures, 30 sputum samples and 30 rectal swabs) of which 10% contained ST175, yielding again 100% sensitivity and specificity. Conclusions The PCR assay developed, showing high sensitivity and specificity for the detection of the ST175 HRC directly from clinical samples, could become a useful tool for guiding infection control and treatment strategies in areas with a high prevalence of this clone.

scholarly journals Detection of Sporothrix schenckii in Clinical Samples by a Nested PCR Assay

2003 ◽  
Vol 41 (4) ◽  
pp. 1414-1418 ◽  
Author(s):  
S. Hu ◽  
W.-H. Chung ◽  
S.-I. Hung ◽  
H.-C. Ho ◽  
Z.-W. Wang ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Sporothrix Schenckii ◽  
Clinical Samples ◽  
Pcr Assay

scholarly journals Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255914
Author(s):  
Monia Ardhaoui ◽  
Emna Ennaifer ◽  
Anna Christina De Matos Salim ◽  
Flávio Marcom Gomez ◽  
Thalja Laasili ◽  
...  
Keyword(s):  
Nested Pcr ◽  
High Sensitivity ◽  
Epidemiological Studies ◽  
Multiple Infection ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Hpv Genotyping ◽  
Genotyping Method ◽  
Specificity And Sensitivity ◽  
Hpv Types

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

A high sensitivity-nested PCR assay for BHV-1 detection in semen of naturally infected bulls

1998 ◽  
Vol 63 (1) ◽  
pp. 1-11 ◽  
Author(s):  
M.A Rocha ◽  
E.F Barbosa ◽  
S.E.F Guimarães ◽  
E Dias Neto ◽  
A.M.G Gouveia
Keyword(s):  
Nested Pcr ◽  
High Sensitivity ◽  
Pcr Assay

scholarly journals Development of A Loop-Mediated Isothermal Amplification (LAMP) Assay for Detection of Relapsing Fever Borreliae

2020 ◽  
Author(s):  
Faezeh Houmansadr ◽  
Mohammad Soleimani ◽  
Saied Reza Naddaf
Keyword(s):  
Nested Pcr ◽  
Extraction Procedure ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Dried Blood Spots ◽  
Negative Control ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Relapsing Fever ◽  
Loop Mediated Isothermal Amplification

Background: This study aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid detec­tion of tick-borne relapsing fever in resource-limited areas. Methods: A set of six primers were designed based on the conserved regions of the Glycerophosphodiester phos­phodiesterase (glpQ) gene of Borrelia species. For sensitivity assay, serial dilutions of a recombinant plasmid contain­ing a 219bp sequence of the glpQ were prepared and used as the template DNA. The LAMP reactions containing the six primers and the reagents required for amplification were incubated at 60–65 °C for 60min in a Loopamp real-time tur­bidimeter. For the specificity test, DNA from 14 other bacteria were included in the assays, and double-distilled water was used as the negative control. Also, DNA from dried blood spots (DBSs) of spirochetemic mice, and blood samples from relapsing fever-suspected patients were examined by the LAMP along a Borrelia-specific nested PCR that targets the rrs-rrl-IGS region. Results: The LAMP detected as low as 90glpQ copies in reactions. The primers reacted with DNA from DBS of spi­rochetemic mice showing spirochete concentrations of ≤ one per a 1000X microscopic field. In clinical samples, the LAMP assay showed a higher sensitivity compared to nested-PCR. The LAMP specificity was 100%, as the primers did not react with other bacteria DNA. Conclusion: The high sensitivity and specificity of the test, along with the simplicity of the DNA extraction procedure, make the LAMP a reliable and adaptable tool for the diagnosis of tick-borne relapsing fever in rural endemic areas.  

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