High-resolution melting analysis for rapid detection of the internationally spreading ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone

2019 ◽  
Vol 75 (1) ◽  
pp. 106-109 ◽  
Author(s):  
Leshan Xiu ◽  
Chi Zhang ◽  
Yamei Li ◽  
Feng Wang ◽  
Junping Peng
Keyword(s):  
High Resolution ◽  
Neisseria Gonorrhoeae ◽  
Internal Control ◽  
High Resolution Melting ◽  
Direct Detection ◽  
High Specificity ◽  
Dual Therapy ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Clinical Samples

Abstract Objectives Increased awareness of the international spread of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone, which threatens recommended dual therapy, is essential. The objective of the present study was to develop and evaluate a rapid, simple and cost-effective method based on high-resolution melting (HRM) analysis for direct detection of the FC428 clone from clinical isolates and specimens. Methods The singleplex HRM assay was designed to identify the FC428 clone by using specific primers, which flank the alteration A311V in the penA-60.001 allele. Analytical performance was initially evaluated by testing 623 isolates and a panel of non-gonococcal strains. To ensure the method can be directly applied in clinical samples, two internal control targets (opa and porA) were also designed and included in the final multiplex HRM assay. Two hundred and eighty-two clinical samples (94 urine and 188 urethral/genital swabs) were then analysed using this multiplex HRM assay. Results The FC428 clone was easily differentiated from the non-mosaic alleles and other mosaic alleles without A311 mutations by comparing the differences in melt curves. Cross-reactivity was not observed for the penA-60.001 allele when testing 15 non-gonococcal Neisseria strains. When applied to the 623 isolates, the HRM assay successfully characterized one isolate as an FC428 clone (MLST1903, NG-MAST3435, NG-STAR233). Our data show that the multiplex HRM assay with high specificity can be directly applied in clinical samples. Conclusions This method can generate results within 90 min at a cost of less than US$0.5 per isolate or sample, making this assay an ideal tool for large epidemiological studies to enhance surveillance of the internationally transmitted ceftriaxone-resistant N. gonorrhoeae FC428 clone.

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

scholarly journals SARS-CoV-2 S1 and N-based serological assays reveal rapid seroconversion and induction of specific antibody response in COVID-19 patients

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
Sharif Hala ◽  
Turki S. Abujamel ◽  
Sawsan S. Alamri ◽  
...  
Keyword(s):  
Antibody Response ◽  
Specific Antibody ◽  
Large Scale ◽  
Disease Onset ◽  
High Specificity ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity ◽  
Human Coronaviruses

Abstract As the Coronavirus Disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses (CoVs). The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

scholarly journals Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

2016 ◽  
Vol 54 (8) ◽  
pp. 2074-2081 ◽  
Author(s):  
Valentina Donà ◽  
Sara Kasraian ◽  
Agnese Lupo ◽  
Yuvia N. Guilarte ◽  
Christoph Hauser ◽  
...  
Keyword(s):  
High Resolution ◽  
Antimicrobial Resistance ◽  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Health Concern ◽  
Content Type ◽  
Resistance To Antibiotics

Resistance to antibiotics used againstNeisseria gonorrhoeaeinfections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences forN. gonorrhoeaeidentification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterizedN. gonorrhoeaestrains, 19 commensalNeisseriaspecies strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identifiedN. gonorrhoeaeand the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcalNeisseriaspecies, and the detection limit was 103to 104genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

scholarly journals SARS-CoV-2 S1 and N-Based Serological Assays Reveal Rapid Seroconversion and Induction of Specific Antibody Response in COVID-19 Patients

2020 ◽  
Author(s):  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
Sherif Hala ◽  
Turki S. Abujamel ◽  
Sawsan S. Alamri ◽  
...  
Keyword(s):  
Antibody Response ◽  
Specific Antibody ◽  
Large Scale ◽  
Disease Onset ◽  
High Specificity ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity ◽  
Human Coronaviruses

As the coronavirus disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses. The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.

scholarly journals Multiple Indirect ELISAs for Serological Detection of SARS-CoV-2 Antibodies

2020 ◽  
Author(s):  
Abdullah Algaissi ◽  
Mohamed A. Alfaleh ◽  
Sherif Hala ◽  
Turki S. Abujamel ◽  
Sawsan S. Alamari ◽  
...  
Keyword(s):  
Antibody Response ◽  
Large Scale ◽  
Disease Onset ◽  
High Specificity ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Tests ◽  
Specificity And Sensitivity ◽  
Novel Coronavirus

As the coronavirus disease 2019 (COVID-19), which is caused by the novel coronavirus SARS-CoV-2, continues to spread rapidly around the world, there is an urgent need for validated serological assays to evaluate viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological tests to study the antibody response in COVID-19 patients. In order to validate the assays, we determined the cut-off values, sensitivity and specificity of the developed assays using sera collected from COVID-19 patients in Saudi Arabia at different time points after disease onset, as well as sera that are seropositive to other human CoVs; namely MERS-CoV, hCoV-OC43, hCoV-NL63, hCoV-229E, and hCoV-HKU1. The SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N) ELISAs that we developed here not only showed high specificity and sensitivity, but also did not show any cross-reactivity with other CoVs. We also showed that all RT-PCR confirmed COVID-19 patients included in our study developed both virus specific IgM and IgG as early as one week after the onset of disease. The availability of these validated assays will enable us to determine the nature and duration of the antibody response mounted in response to SARS-CoV-2 infection. It will also allow conducting large-scale epidemiological studies to determine evidence of previous exposure to the virus and assess the true extent of virus spread within communities.

scholarly journals DMSO Increases Mutation Scanning Detection Sensitivity of High-Resolution Melting in Clinical Samples

2015 ◽  
Vol 61 (11) ◽  
pp. 1354-1362 ◽  
Author(s):  
Chen Song ◽  
Elena Castellanos-Rizaldos ◽  
Rafael Bejar ◽  
Benjamin L Ebert ◽  
G Mike Makrigiorgos
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Digital Pcr ◽  
Detection Sensitivity ◽  
Clinical Samples ◽  
Conventional Pcr ◽  
Cross Hybridization ◽  
Mutation Scanning ◽  
Extra Step ◽  
Clinically Significant

Abstract BACKGROUND Mutation scanning provides the simplest, lowest-cost method for identifying DNA variations on single PCR amplicons, and it may be performed before sequencing to avoid screening of noninformative wild-type samples. High-resolution melting (HRM) is the most commonly used method for mutation scanning. With PCR-HRM, however, mutations less abundant than approximately 3%–10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application. METHODS We used serial dilution of cell lines containing the TP53 exon 8 mutation to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted coamplification at lower denaturation temperature (COLD)-PCR with an extra step for cross-hybridization, followed by preferential denaturation and amplification at optimized critical temperature (full-COLD-PCR), to further enrich low-level mutations before HRM with or without DMSO, and we used droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional PCR-HRM and full-COLD-PCR-HRM with and without DMSO were used for mutation scanning of TP53 exon 8 in cancer samples containing known mutations and myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined. RESULTS The detection sensitivity of PCR-HRM scanning increases 2- to 5-fold in the presence of DMSO, depending on mutation type and sequence context, and can typically detect mutation abundance of approximately 1%. When mutation enrichment is applied during amplification with full-COLD-PCR followed by HRM in the presence of DMSO, mutations with 0.2%–0.3% abundance in TP53 exon 8 can be detected. CONCLUSIONS DMSO improves HRM mutation scanning sensitivity with saturating dyes. When full-COLD-PCR is used, followed by DMSO-HRM, the overall improvement is about 20-fold compared with conventional PCR-HRM.

scholarly journals Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies

2017 ◽  
Vol 63 (10) ◽  
pp. 1605-1613 ◽  
Author(s):  
Ioannis Ladas ◽  
Mariana Fitarelli-Kiehl ◽  
Chen Song ◽  
Viktor A Adalsteinsson ◽  
Heather A Parsons ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Wild Type ◽  
Liquid Biopsies ◽  
Targeted Resequencing ◽  
Double Stranded Dna ◽  
Closed Tube

Abstract BACKGROUND The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing. METHODS We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. RESULTS Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10–20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. CONCLUSIONS Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing.

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