scholarly journals Single-tube, dual channel pentaplexing for the identification of Candida strains associated with human infection

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mohd Hanif Jainlabdin ◽  
Ambalika Batra ◽  
Edith Sánchez Paredes ◽  
Francisca Hernández Hernández ◽  
Guoliang Fu ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Fungal Infections ◽  
Melting Curve ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Melting Curve Analysis ◽  
Detection Range ◽  
Single Tube ◽  
Dual Channel ◽  
Specificity And Sensitivity

Abstract Invasive candidiasis is one of the most common nosocomial fungal infections worldwide. Delayed implementation of effective antifungal treatment caused by inefficient Candida diagnosis contributes to its notoriously high mortality rates. The availability of better Candida diagnostic tools would positively impact patient outcomes. Here, we report on the development of a single-tube, dual channel pentaplex molecular diagnostic assay based on Multiplex Probe Amplification (MPA) technology. It allows simultaneous identification of C. auris, C. glabrata and C. krusei, at species-level as well as of six additional albicans and non-albicans pathogenic Candida at genus level. The assay overcomes the one-channel one-biomarker limitation of qPCR-based assays. Assay specificities are conferred by unique biomarker probe pairs with characteristic melting temperatures; post-amplification melting curve analysis allows simple identification of the infectious agent. Alerting for the presence of C. auris, the well-characterised multi-drug resistant outbreak strain, will facilitate informed therapy decisions and aid antifungal stewardship. The MPA-Candida assay can also be coupled to a pan-Fungal assay when differentiation between fungal and bacterial infections might be desirable. Its multiplexing capacity, detection range, specificity and sensitivity suggest the potential use of this novel MPA-Candida assay in clinical diagnosis and in the control and management of hospital outbreaks.

scholarly journals A High Resolution DNA Melting Curve Analysis for the Rapid and Efficient Molecular Diagnostics of Extended Spectrum β-Lactamase Determinants from Foodborne Escherichia coli

2020 ◽  
Vol 8 (1) ◽  
pp. 90 ◽  
Author(s):  
Patrick Murigu Kamau Njage ◽  
Elna Buys
Keyword(s):  
Melting Curve ◽  
Temperature Shift ◽  
Molecular Diagnostic ◽  
Melting Curve Analysis ◽  
Phylogenetic Groups ◽  
Accurate Identification ◽  
Extended Spectrum ◽  
Multiplex Pcr Assay ◽  
Specificity And Sensitivity ◽  
Closed Tube

The accurate identification of Extended-Spectrum β-Lactamase (ESBL) genes in Gram-negative bacteria is necessary for surveillance and epidemiological studies of transmission through foods. We report a novel rapid, cheap, and accurate closed tube molecular diagnostic tool based on two multiplex HRM protocols for analysis of the predominant ESBL families encountered in foods. The first multiplex PCR assay targeted blaCTX-M including phylogenetic groups 1 (CTX-M-1-15, including CTX-M-1, CTX-M-3 and CTX-M-15), 2 (CTX-M-2), and 9 (CTX-M-9-14, including CTX-M-9 and CTX-M-14). The second assay involved blaTEM /bla CTX-M /blaSHV, including TEM variants (TEM-1 and TEM-2), SHV-1-56 (SHV-1, SHV-2 and SHV-56), and CTX-M-8-41 (CTX-M-8, CTX-M-25, CTX-M-26 and CTX-M-39 to CTX-M-41). The individual melting curves were differentiated by a temperature shift according to the type of ESBL gene. The specificity and sensitivity of the first assay were 100% and 98%, respectively. For the second assay, the specificity and sensitivity were 87% and 89%, respectively. The detection of ESBL variants or mutations in existing genes was also demonstrated by the subtyping of a variant of the CTXM-1-15. The HRM is a potential tool for the rapid detection of present β-lactamase genes and their characterization in a highly sensitive, closed-tube, inexpensive method that is applicable in high throughput studies.

scholarly journals Same-Day Simultaneous Diagnosis of Bacterial and Fungal Infections in Clinical Practice by Nanopore Targeted Sequencing

2020 ◽  
Author(s):  
Ming Wang ◽  
Aisi Fu ◽  
Ben Hu ◽  
Gaigai Shen ◽  
Ran Liu ◽  
...  
Keyword(s):  
Clinical Practice ◽  
Bacterial Infections ◽  
Fungal Infections ◽  
Culture Method ◽  
Targeted Sequencing ◽  
Therapeutic Monitoring ◽  
Rrna Gene ◽  
Culture Methods ◽  
Detection Range ◽  
Sequencing Platform

AbstractBACKGROUNDAs approximately 19% of global deaths are attributable to infectious diseases, early diagnosis of infection is very important to reduce mortality. Traditional infection detection strategies have limited sensitivity, detection range, and turnaround times; a detection technology that can simultaneously detect bacterial and fungal infections within 24 h is urgently need in clinical settings.METHODSWe developed nanopore targeted sequencing (NTS) for same-day simultaneous Diagnosis of fungal and bacterial infections. NTS was developed by amplification of 16s rRNA gene (for bacteria), IST1/2 gene (for fungal), and rpoB (for Mycobacterium spp.) using multiple primers, and sequenced by a real-time nanopore sequencing platform. An in-house bioinformatic analyze pipeline was used to diagnose the infectious pathogens by mapping the sequencing results with the constructed databases.RESULTSComparison of 1312 specimens from 1257 patients using NTS and culture method; NTS detected pathogens in 58.71% of specimens from patients, compared to 22.09% detected using the culture method. NTS showed significantly higher sensitivity than culture methods for many pathogens. Importantly, a turnaround time of <24 h for all specimens, and a pre-report within 6 h in emergency cases was possible in clinical practice. Modification of antibiotic therapy and maintenance of original anti-infection regimens in 51.52% (17/33) and 36.36% (12/33) of patients was in accordance with NTS results, and quantitative monitoring of clinical treatment effects was evaluated in four patients by continuous NTS tests.CONCLUSIONSApplication of NTS in clinically detected pathogens can improve targeted antibiotic treatment and therapeutic monitoring.

scholarly journals Rapid Detection of ERG11-Associated Azole Resistance and FKS-Associated Echinocandin Resistance in Candida auris

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Xin Hou ◽  
Annie Lee ◽  
Cristina Jiménez-Ortigosa ◽  
Milena Kordalewska ◽  
David S. Perlin ◽  
...  
Keyword(s):  
Rapid Assessment ◽  
Melting Curve ◽  
Rapid Identification ◽  
Molecular Diagnostic ◽  
Melting Curve Analysis ◽  
Azole Resistance ◽  
Candida Auris ◽  
Content Type ◽  
Echinocandin Resistance

ABSTRACT Candida auris is an emerging multidrug-resistant yeast that can cause serious invasive infections. The accurate and rapid assessment of antifungal resistance is important for effective patient management. A novel and highly accurate diagnostic platform was established for the rapid identification of ERG11 mutations conferring azole resistance and FKS1 mutations associated with echinocandin resistance in C. auris. Using allele-specific molecular beacons and DNA melting curve analysis following asymmetric PCR, a duplex ERG11 assay and a simplex FKS1 HS1 assay were developed to identify the most prominent resistance-associated mutations (Y132F and K143R in ERG11; S639F in FKS1 HS1) within 2 h. Assays were validated by testing a panel of 94 C. auris clinical isolates in a blind manner. The molecular diagnostic results from the assays were 100% concordant with DNA sequencing results. This platform has the potential to overcome the deficiencies of existing in vitro susceptibility-based assays to identify azole- and/or echinocandin-resistant C. auris, and thus, it holds promise as a surrogate diagnostic method to direct antifungal therapy more effectively.

scholarly journals Siderophore-Based Molecular Imaging of Fungal and Bacterial Infections—Current Status and Future Perspectives

2020 ◽  
Vol 6 (2) ◽  
pp. 73 ◽  
Author(s):  
Milos Petrik ◽  
Joachim Pfister ◽  
Matthias Misslinger ◽  
Clemens Decristoforo ◽  
Hubertus Haas
Keyword(s):  
Molecular Imaging ◽  
Bacterial Infections ◽  
Fluorescent Dyes ◽  
Fungal Infections ◽  
Current Status ◽  
Specificity And Sensitivity ◽  
Positron Emission ◽  
Imaging Probes ◽  
Triacetylfusarinine C ◽  
Gallium 68

Invasive fungal infections such as aspergillosis are life-threatening diseases mainly affecting immuno-compromised patients. The diagnosis of fungal infections is difficult, lacking specificity and sensitivity. This review covers findings on the preclinical use of siderophores for the molecular imaging of infections. Siderophores are low molecular mass chelators produced by bacteria and fungi to scavenge the essential metal iron. Replacing iron in siderophores by radionuclides such as gallium-68 allowed the targeted imaging of infection by positron emission tomography (PET). The proof of principle was the imaging of pulmonary Aspergillus fumigatus infection using [68Ga]Ga-triacetylfusarinine C. Recently, this approach was expanded to imaging of bacterial infections, i.e., with Pseudomonas aeruginosa. Moreover, the conjugation of siderophores and fluorescent dyes enabled the generation of hybrid imaging compounds, allowing the combination of PET and optical imaging. Nevertheless, the high potential of these imaging probes still awaits translation into clinics.

DETECTION OF NOTCH1 c.7544_7545delCT MUTATION IN CHRONIC LYMPHOCYTIC LEUKEMIA USING CONVENTIONAL AND REAL-TIME POLYMERASE CHAIN REACTION

2016 ◽  
Vol 38 (2) ◽  
pp. 112-116 ◽  
Author(s):  
N I Bilous ◽  
I V Abramenko ◽  
A A Chumak ◽  
I S Dyagil ◽  
Z V Martina
Keyword(s):  
Polymerase Chain Reaction ◽  
Chronic Lymphocytic Leukemia ◽  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Lymphocytic Leukemia ◽  
Melting Curve Analysis ◽  
Chain Reaction ◽  
Specificity And Sensitivity ◽  
Polymerase Chain

Aim: To evaluate real-time polymerase chain reaction (PCR) assay system for detection of NOTCH1 c.7541_754delCT mutation in chronic lymphocytic leukemia (CLL) patients. Material and Methods: A total of 325 CLL patients were included in the study. Screening for NOTCH1 c.7544_7545delCT was performed using conventional PCR-based amplification refractory mutation system (ARMS) method. All 33 samples harboring c.7544_7545delCT allele and 5 negative cases as control were submitted to real-time PCR. Results: Specificity and sensitivity of two PCR techniques were comparable. NOTCH1 c.7544_7545delCT mutation was found by ARMS in 10.1% of CLL patients, which is consistent with the data of other studies. However, the results of ARMS PCR in a minority of cases (2.15%) were doubtful and required reinvestigation. Real-time PCR, being less time-consuming, showed advantage in the assessment of the amplification’s specificity (using the melting curve analysis). It also allows the quantitative assessment of NOTCH1-mutated clone. Conclusion: NOTCH1 c.7544_7545delCT mutation resulting in removal of the C-terminal PEST domain, deregulation of NOTCH1-dependent signaling pathways, has negative influence on prognosis of CLL and efficiency of therapy with anti-CD20 monoclonal antibodies. Real-time PCR allows the fast and reliable detection of c.7544_7545delCT mutation and can be used for the screening of this molecular lesion in CLL patients.

Use of Two Reporter Dyes without Interference in a Single-Tube Rapid-Cycle PCR: α1-Antitrypsin Genotyping by Multiplex Real-Time Fluorescence PCR with the LightCycler

2000 ◽  
Vol 46 (2) ◽  
pp. 156-161 ◽  
Author(s):  
Nicolas von Ahsen ◽  
Michael Oellerich ◽  
Ekkehard Schütz
Keyword(s):  
Mutation Detection ◽  
Fluorescent Dyes ◽  
Simultaneous Detection ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Single Tube ◽  
Antitrypsin Deficiency ◽  
Color Compensation ◽  
Assay Time ◽  
Pcr Method

Abstract Background: α1-Antitrypsin is the major plasma serine protease inhibitor. Its deficiency is mainly associated with the alleles PI*S and PI*Z and can lead to obstructive lung disease in adults and to liver cirrhosis during childhood. Methods: A multiplex PCR method has been established that uses two sets of primers to amplify the gene regions covering the PI*S or PI*Z mutations sites. Mutation detection was performed on the LightCycler by melting curve analysis of detection probes labeled with two different fluorescent dyes, LC-Red640 and LC-Red705. Results: Unequivocal genotyping results were obtained for all investigated samples in an assay time of ∼30 min. The color compensation procedure greatly improved the readability of the resulting diagnostic melting curves. Conclusions: To our knowledge, this is the first report of simultaneous detection of two mutations in a single tube by PCR of genomic DNA and the use of two different reporter dyes with the LightCycler color compensation feature. This approach is a rapid, convenient, and economic alternative to other methods described to date for the detection of α1-antitrypsin deficiency alleles.

scholarly journals New Panfungal Real-Time PCR Assay for Diagnosis of Invasive Fungal Infections

2016 ◽  
Vol 54 (12) ◽  
pp. 2910-2918 ◽  
Author(s):  
Clara Valero ◽  
Laura de la Cruz-Villar ◽  
Óscar Zaragoza ◽  
María José Buitrago
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fungal Infections ◽  
Melting Curve ◽  
Fungal Species ◽  
Invasive Fungal Infections ◽  
Clinical Samples ◽  
Melting Curve Analysis ◽  
Pcr Assay ◽  
Suitable Alternative

The diagnosis of invasive fungal infections (IFIs) is usually based on the isolation of the fungus in culture and histopathological techniques. However, these methods have many limitations often delaying the definitive diagnosis. In recent years, molecular diagnostics methods have emerged as a suitable alternative for IFI diagnosis. When there is not a clear suspicion of the fungus involved in the IFI, panfungal real-time PCR assays have been used, allowing amplification of any fungal DNA. However, this approach requires subsequent amplicon sequencing to identify the fungal species involved, increasing response time. In this work, a new panfungal real-time PCR assay using the combination of an intercalating dye and sequence-specific probes was developed. After DNA amplification, a melting curve analysis was also performed. The technique was standardized by using 11 different fungal species and validated in 60 clinical samples from patients with proven and probable IFI. A melting curve database was constructed by collecting those melting curves obtained from fungal species included in the standardization assay. Results showed high reproducibility (coefficient of variation [CV] < 5%; r > 0.95) and specificity (100%). The overall sensitivity of the technique was 83.3%, with the group of fungi involved in the infection detected in 77.8% of the positive samples with IFIs covered by molecular beacon probes. Moreover, sequencing was avoided in 67.8% of these “probe-positive” results, enabling report of a positive result in 24 h. This technique is fast, sensitive, and specific and promises to be useful for improving early diagnosis of IFIs.

scholarly journals Comparison of bluetongue virus detection and quantitation methods in south India

2014 ◽  
Vol 8 (10) ◽  
pp. 1307-1312
Author(s):  
Subhra Subhadra ◽  
Subrat Kumar ◽  
Veluvarthy VS Suryanarayana ◽  
Daggupati Sreenivasulu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Disease Diagnosis ◽  
Sybr Green ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Animal Blood ◽  
Specificity And Sensitivity

Introduction: Bluetongue (BT), a vector-borne viral disease, primarily affects sheep. Of the 26 serotypes of BTV identified so far, 22 are reported to be circulating in India. Due to an increase in vector population and delays in disease diagnosis, the BT control program heavily relies on rapid and confirmatory diagnosis. Polymerase chain reaction (PCR)-based real-time detection assays may be an ideal method to detect the BTV genome in animal blood at an early stage of infection. Methodology: In this study, a SYBR green-based real-time RT-PCR assay was evaluated, validated, and compared with conventional RT-PCR. The specificity and sensitivity of an assay using BTV-2 RNA extracted from tenfold serially diluted (starting from 1.0 TCID50/mL) cell culture virus was also evaluated. Results: While conventional RT-PCR could detect 3.16×102 TCID50 of virus/mL, the real-time PCR test had a detection limit of 3.16×10-4 TCID50/mL. Melting curve analysis indicated the absence of non-specific amplification (R2 = 0.987). Out of the 32 infected blood samples examined, 24 tested positive for BTV RNA. Seven that were found negative through conventional PCR tested positive through real-time PCR. Conclusions: These results showed that the SYBR green-based real-time PCR assay is rapid, sensitive, and equally specific in the diagnosis of BT in BTV-affected animals.

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