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.

scholarly journals Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovorain Asymptomatic Plant Material

2000 ◽  
Vol 66 (5) ◽  
pp. 2071-2078 ◽  
Author(s):  
Pablo Llop ◽  
Anna Bonaterra ◽  
Javier Peñalver ◽  
María M. López
Keyword(s):  
Nested Pcr ◽  
Plant Material ◽  
High Sensitivity ◽  
Epidemiological Studies ◽  
Sample Volume ◽  
Small Sample ◽  
Asymptomatic Plant ◽  
Specificity And Sensitivity ◽  
Closed Tube ◽  
Pure Cultures

ABSTRACT A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.

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 Sensitive and Specific Detection of Platelet-Derived and Tissue Factor–Positive Extracellular Vesicles in Plasma Using Solid-Phase Proximity Ligation Assay

TH Open ◽  
2018 ◽  
Vol 02 (03) ◽  
pp. e250-e260 ◽  
Author(s):  
Åsa Thulin ◽  
Junhong Yan ◽  
Mikael Åberg ◽  
Christina Christersson ◽  
Masood Kamali-Moghaddam ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Extracellular Vesicles ◽  
Solid Phase ◽  
Flow Cytometric Analysis ◽  
High Sensitivity ◽  
Small Sample ◽  
Proximity Ligation Assay ◽  
Clinical Samples ◽  
Proximity Ligation ◽  
Specificity And Sensitivity

AbstractExtracellular vesicles (EVs) derived from blood cells are promising biomarkers for various diseases. However, they are difficult to measure accurately in plasma due to their small size. Here, we demonstrate that platelet-derived EVs in plasma can be measured using solid-phase proximity ligation assay with high sensitivity and specificity using very small sample volume of biological materials. The results correlate well with high-sensitivity flow cytometry with the difference that the smallest EVs are detected. Briefly, the EVs are first captured on a solid phase, using lactadherin binding, and detection requires recognition with two antibodies followed by qPCR. The assay, using cholera toxin subunit-B or lactadherin as capture agents, also allowed detection of the more rare population of tissue factor (TF)-positive EVs at a concentration similar to sensitive TF activity assays. Thus, this assay can detect different types of EVs with high specificity and sensitivity, and has the potential to be an attractive alternative to flow cytometric analysis of preclinical and clinical samples. Improved techniques for measuring EVs in plasma will hopefully contribute to the understanding of their role in several diseases.

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 A Novel Xenonucleic Acid Mediated Molecular Clamping Technology for Early Colorectal Cancer Screening

2020 ◽  
Author(s):  
Qing Sun ◽  
Larry Pastor ◽  
Jinwei Du ◽  
Michael J. Powell ◽  
Aiguo Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Precancerous Lesions ◽  
Cancer Diagnostics ◽  
Clinical Samples ◽  
Early Colorectal Cancer ◽  
Analytical Sensitivity ◽  
Assay Sensitivity ◽  
Specificity And Sensitivity ◽  
Ffpe Samples ◽  
Total Dna

Abstract BackgroundColorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape TM for early colorectal cancer diagnostics. MethodsNineteen mutations in four genes APC, KRAS, BRAF and CTNNB1 associated with early events in CRC pathogenesis are targeted in the ColoScapeTM assay. Xenonucleic Acid (XNA) mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and a ROC cure was applied to evaluate its performance.ResultsThe data showed that the assay analytical sensitivity is 0.5% Variant Allele Frequency, corresponding to ~7-8 copies of mutant DNA with 5ng total DNA input per test. This assay is highly reproducible with intra-assay CV <3% and inter-assay <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were 100% (95% CI, 80.3-97.5%) and 92.2% (95% CI, 94.7-100%) respectively with AUC being about 0.96; and 96% (95% CI, 77.6-99.7%) specificity and 92% (95% CI, 86.1-95.6%) sensitivity with AUC 0.94 for CRC FFPE; and 95% specificity (95% CI, 82.5%-99.1%) and 62.5% sensitivity (95% CI, 35.8%-83.7%) with AUC 0.79 for precancerous lesions cfDNA.ConclusionsXNA mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

scholarly journals A novel xenonucleic acid-mediated molecular clamping technology for early colorectal cancer screening

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0244332
Author(s):  
Qing Sun ◽  
Larry Pastor ◽  
Jinwei Du ◽  
Michael J. Powell ◽  
Aiguo Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Precancerous Lesions ◽  
Cancer Diagnostics ◽  
Clinical Samples ◽  
Early Colorectal Cancer ◽  
Analytical Sensitivity ◽  
Assay Sensitivity ◽  
Specificity And Sensitivity ◽  
Ffpe Samples ◽  
Total Dna

Background Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape TM assay for early colorectal cancer diagnostics. Methods Nineteen mutations in four genes (APC, KRAS, BRAF and CTNNB1) associated with early events in CRC pathogenesis are targeted in the ColoScapeTM assay. Xenonucleic Acid (XNA)-mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and an ROC curve was applied to evaluate its performance. Results The data showed that the assay analytical sensitivity was 0.5% Variant Allele Frequency, corresponding to ~7–8 copies of mutant DNA with 5 ng total DNA input per test. This assay is highly reproducible with intra-assay CV of <3% and inter-assay CV of <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were: 100% (95% CI, 80.3–97.5%) and 92.2% (95% CI, 94.7–100%), respectively, with AUC of 0.96; 96% specificity (95% CI, 77.6–99.7%) and 92% sensitivity (95% CI, 86.1–95.6%) with AUC of 0.94 for CRC FFPE; 95% specificity (95% CI, 82.5%–99.1%) and 62.5% sensitivity (95% CI, 35.8%–83.7%) with AUC of 0.79 for precancerous lesions cfDNA. Conclusions The XNA-mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

scholarly journals Q Fever in the Greek Island of Crete: Detection, Isolation, and Molecular Identification of Eight Strains of Coxiella burnetii from Clinical Samples

1998 ◽  
Vol 36 (7) ◽  
pp. 2063-2067 ◽  
Author(s):  
Ioanna Spyridaki ◽  
Achilleas Gikas ◽  
Diamantis Kofteridis ◽  
Anna Psaroulaki ◽  
Yannis Tselentis
Keyword(s):  
Coxiella Burnetii ◽  
Nested Pcr ◽  
Q Fever ◽  
Buffy Coat ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Antibody Technique ◽  
Presenting Symptoms ◽  
Specificity And Sensitivity ◽  
Reference Strains

Over a period of 6 years (1989 to 1995), serum samples from 3,300 patients suspected to be infected by Coxiella burnetii were assayed for the presence of antibodies against antigen phase II of the microorganism by the indirect immunofluorescence antibody technique (IFAT). One hundred fifty-two cases were recorded, and blood samples from 17 patients were cultured for the isolation of the pathogen. By a centrifugation shell vial technique, eight strains were isolated from patients suffering from acute Q fever. The microorganism was detected in the cultures by IFAT, by Gimenez staining, and by the cytopathogenic effect on Vero and human embryonic lung (HEL) cells. PCR followed by restriction fragment length polymorphism analysis was used to confirm the diagnosis and identify the Coxiella burnetii strains within the cell cultures as well as to compare them with reference strains. In order to avoid time-consuming cultures, to achieve direct detection of Coxiella burnetii in clinical samples (blood, buffy coat, etc.), and to increase the specificity and sensitivity of the detection, nested PCR was performed. The first step of DNA extraction was performed with the QIAamp blood kit 250. For the second step of the PCR assays, the conditions of temperature and times of recycling were properly modified, and the microorganism was detected within 4 h. Our study demonstrates that Q fever is an endemic disease in Crete and that the diagnosis of Coxiella burnetii infection can be rapidly achieved by the detection of the microorganism in buffy coat samples by nested PCR. Although the presenting symptoms of the disease in this study differed from those in other studies, the Cretan strains do not differ genotypically from the reference strains (Nine Mile and Q212).

scholarly journals A Novel Xenonucleic Acid Mediated Molecular Clamping Technology for Early Colorectal Cancer Screening

2020 ◽  
Author(s):  
Qing Sun ◽  
Larry Pastor ◽  
Jinwei Du ◽  
Michael J. Powell ◽  
Aiguo Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Precancerous Lesions ◽  
Cancer Diagnostics ◽  
Clinical Samples ◽  
Early Colorectal Cancer ◽  
Analytical Sensitivity ◽  
Assay Sensitivity ◽  
Specificity And Sensitivity ◽  
Ffpe Samples ◽  
Total Dna

ABSTRACTBackgroundColorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape ™ for early colorectal cancer diagnostics.MethodsNineteen mutations in four genes APC, KRAS, BRAF and CTNNB1 associated with early events in CRC pathogenesis are targeted in the ColoScape™ assay. Xenonucleic Acid (XNA) mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and a ROC cure was applied to evaluate its performance.ResultsThe data showed that the assay analytical sensitivity is 0.5% Variant Allele Frequency, corresponding to ~7-8 copies of mutant DNA with 5ng total DNA input per test. This assay is highly reproducible with intra-assay CV <3% and inter-assay <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were 100% (95% CI, 80.3-97.5%) and 92.2% (95% CI, 94.7-100%) respectively with AUC being about 0.96; and 96% (95% CI, 77.6-99.7%) specificity and 92% (95% CI, 86.1-95.6%) sensitivity with AUC 0.94 for CRC FFPE; and 95% specificity (95% CI, 82.5%-99.1%) and 62.5% sensitivity (95% CI, 35.8%-83.7%) with AUC 0.79 for precancerous lesions cfDNA.ConclusionsXNA mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

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.  

A novel xenonucleic acid mediated molecular clamping technology for early colorectal cancer diagnostics.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e16106-e16106
Author(s):  
Qing Sun ◽  
Larry Pastor ◽  
Jinwei Du ◽  
Michael Joseph Powell ◽  
Aiguo Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clinical Performance ◽  
Cancer Diagnostics ◽  
Clinical Samples ◽  
Early Colorectal Cancer ◽  
Analytical Sensitivity ◽  
Assay Sensitivity ◽  
Specificity And Sensitivity ◽  
Ffpe Samples ◽  
Performance Results

e16106 Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called ColoScape for early colorectal cancer diagnostics. Methods: Nineteen mutations in four genes APC, KRAS, BRAF and CTNNB1 associated with early events in CRC pathogenesis are targeted in the ColoScape assay. Xenonucleic Acid (XNA) mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were assessed for clinical performance, and a ROC curve was applied to evaluate its performance. Results: The data showed that the assay analytical sensitivity is 0.5% Variant Allele Frequency, corresponding to ~7-8 copies of mutant DNA with 5ng total DNA input per test. This assay is highly reproducible with intra-assay CV < 3% and inter-assay CV < 5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with different stages of CRC. The preliminary assay clinical specificity and sensitivity for cfDNA were 100% (95% CI, 80.3-97.5%) and 92.2% (95% CI, 94.7-100%) respectively with AUC being about 0.96; and 96% (95% CI, 77.6-99.7%) specificity and 92% (95% CI, 86.1-95.6%) sensitivity with AUC 0.94 for FFPE. Conclusions: XNA mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of CRC mutations in cfDNA or FFPE samples.

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