scholarly journals Development a hydrolysis probe-based quantitative PCR assay for the specific detection and quantification of Candida auris

2020 ◽  
Author(s):  
Hadis Jafarian ◽  
Hossein Khodadadi ◽  
Parisa Badiee
Keyword(s):  
Real Time ◽  
Multidrug Resistant ◽  
Qpcr Assay ◽  
Nuclear Ribosomal Dna ◽  
Candida Auris ◽  
Accurate Identification ◽  
Hydrolysis Probe ◽  
Standard Curve ◽  
Specific Pcr ◽  
Species Specific

Background and Purpose: Candida auris is an emerging multidrug-resistant pathogen. The identification of this species with the conventional phenotypic or biochemical mycological methods may lead to misidentification. Molecular-based species-specific identification methods such as quantitative real-time polymerase chain reaction (qPCR) facilitate a more reliable identification of C. auris than mycological methods. Regarding this, the present study aimed to develop a hydrolysis probe-based qPCR assay for the rapid, accurate identification of C. auris. Materials and Methods: The internal transcribed spacer 2 regions in the nuclear ribosomal DNA of C. auris and other related yeasts were assayed to find a specific PCR target for C. auris. A 123-base-pair target was selected, and primers and a probe were designed for hydrolysis probe-based real-time PCR with TaqMan chemistry. Ten-fold serial dilutions of C. auris ranging from 106 to 100 CFU/mL were prepared to establish a standard curve to quantify the yeast. Results: The qPCR assay was able to identify and quantify C. auris with a detection limit of 1 C. auris CFU per reaction. Specificity was confirmed by the non-amplification of the sequences belonging to other Candida species, yeasts, molds, bacteria, or human DNAs. The standard curve of the assay showed a highly significant linearity between threshold values and dilution rates (R2=0.99; slope=−3.42). Conclusion: The applied qPCR assay facilitated the rapid and accurate identification and quantification of emerging opportunistic C. auris. Therefore, considering the promising test validation results, we succeeded to develop a rapid and accurate hydrolysis probe-based qPCR assay for the screening and identification of C. auris.

scholarly journals Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

2021 ◽  
Vol 11 ◽  
Author(s):  
Reza Fotouhi-Ardakani ◽  
Seyedeh Maryam Ghafari ◽  
Paul Donald Ready ◽  
Parviz Parvizi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Major ◽  
Taqman Probe ◽  
Amino Acid Permease ◽  
Specific Primers ◽  
Accurate Identification ◽  
Parasitic Load ◽  
Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

A high‐throughput and rapid method for accurate identification of emerging multidrug‐resistant Candida auris

Mycoses ◽  
2019 ◽  
Vol 62 (6) ◽  
pp. 513-518 ◽  
Author(s):  
Ausaf Ahmad ◽  
Jonathan E. Spencer ◽  
Shawn R. Lockhart ◽  
Sabrina Singleton ◽  
David J. Petway ◽  
...  
Keyword(s):  
Rapid Method ◽  
High Throughput ◽  
Multidrug Resistant ◽  
Candida Auris ◽  
Accurate Identification

scholarly journals Species-Specific PCR as a Tool for the Identification of Burkholderia gladioli

2000 ◽  
Vol 38 (1) ◽  
pp. 282-285
Author(s):  
Paul W. Whitby ◽  
Lauren C. Pope ◽  
Karen B. Carter ◽  
John J. LiPuma ◽  
Terrence L. Stull
Keyword(s):  
Cystic Fibrosis ◽  
Sensitivity And Specificity ◽  
Burkholderia Cepacia ◽  
Bacterial Species ◽  
23S Rrna ◽  
Computer Assisted ◽  
Accurate Identification ◽  
Specific Pcr ◽  
Burkholderia Gladioli ◽  
Species Specific

ABSTRACT Burkholderia gladioli colonizes the respiratory tracts of patients with cystic fibrosis and chronic granulomatous disease. However, due to the high degree of phenotypic similarity between this species and closely related species in the Burkholderia cepacia complex, accurate identification is difficult. Incorrect identification of these species may have serious repercussions for the management of patients with cystic fibrosis. To develop an accurate procedure for the identification of B. gladioli , a molecular method to discriminate between this species and other species commonly isolated from the sputa of patients with cystic fibrosis was investigated. The 23S ribosomal DNA was cloned from several clinical isolates of B. gladioli , and the nucleotide sequence was determined. Computer-assisted sequence comparisons indicated four regions of the 23S rRNA specific for this species; these regions were used to design three primer pairs for species-specific PCR. Two of the primer pairs showed 100% sensitivity and specificity for B. gladioli when tested against a panel of 47 isolates comprising 19 B. gladioli isolates and 28 isolates of 16 other bacterial species. One of the primer pairs was further assessed for species specificity by using a panel of 102 isolates obtained from the Burkholderia cepacia Research Laboratory and Repository. The species-specific PCR was positive for 70 of 74 isolates of B. gladioli and was negative for all other bacterial species examined. Overall, this primer pair displayed a sensitivity and specificity of 96% (89 of 93) and 100%, respectively. These data demonstrate the potential of species-specific PCR for the identification of B. gladioli .

scholarly journals Rapid and Accurate Molecular Identification of the Emerging Multidrug-Resistant Pathogen Candida auris

2017 ◽  
Vol 55 (8) ◽  
pp. 2445-2452 ◽  
Author(s):  
Milena Kordalewska ◽  
Yanan Zhao ◽  
Shawn R. Lockhart ◽  
Anuradha Chowdhary ◽  
Indira Berrio ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Related Species ◽  
Multidrug Resistant ◽  
Candida Auris ◽  
Content Type ◽  
Invasive Infections ◽  
Candida Lusitaniae ◽  
Pcr Assays ◽  
Phenotypic Tests

ABSTRACT Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii , Candida haemulonii , and Candida lusitaniae . Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species.

scholarly journals Real-Time PCR for Detection of NDM-1 Carbapenemase Genes from Spiked Stool Samples

2011 ◽  
Vol 55 (9) ◽  
pp. 4038-4043 ◽  
Author(s):  
Thierry Naas ◽  
Ayla Ergani ◽  
Amélie Carrër ◽  
Patrice Nordmann
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Multidrug Resistant ◽  
Qpcr Assay ◽  
Resistant Bacteria ◽  
Amplification Efficiency ◽  
Bacterial Isolates ◽  
Limits Of Detection ◽  
Stool Samples ◽  
Automated Dna Extraction

ABSTRACTAn in-house quantitative real-time PCR (qPCR) assay using TaqMan chemistry has been developed to detect NDM-1 carbapenemase genes from bacterial isolates and directly from stool samples. The qPCR amplification ofblaNDM-1DNA was linear over 10 log dilutions (r2= 0.99), and the amplification efficiency was 1.03. The qPCR detection limit was reproducibly 1 CFU, or 10 plasmid molecules, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria harboring other β-lactam resistance genes. Feces spiked with decreasing amounts of enterobacterial isolates producing NDM-1 were spread on ChromID ESBL and on CHROMagar KPC media and were subjected to the qPCR. The limits of carbapenem-resistant bacterial detection from stools was reproducibly 1 × 101to 3 × 101CFU/100 mg feces with ChromID ESBL medium. The CHROMagar KPC culture medium had higher limits of detection (1 × 101to 4 × 103CFU/ml), especially with bacterial isolates having low carbapenem MICs. The limits of detection with the qPCR assay were reproducibly below 1 × 101CFU/100 mg of feces by qPCR assay. Samples spiked with NDM-1-negative bacteria were negative by qPCR. The sensitivity and specificity of theblaNDM-1qPCR assay on spiked samples were 100% in both cases. Using an automated DNA extraction system (QIAcube system), the qPCR assay was reproducible. The use of qPCR is likely to shorten the time forblaNDM-1detection from 48 h to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.

scholarly journals Real-Time PCR for Detection and Identification of Anguina funesta, A. agrostis, A. tritici, and A. pacificae

Plant Disease ◽  
2015 ◽  
Vol 99 (11) ◽  
pp. 1584-1589 ◽  
Author(s):  
Wenbin Li ◽  
Zonghe Yan ◽  
Mark K. Nakhla ◽  
Andrea M. Skantar
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
The United States ◽  
Grass Species ◽  
Specific Primers ◽  
Accurate Identification ◽  
Detection And Identification ◽  
Stem Gall ◽  
Species Specific

A number of seed, leaf, and stem gall nematodes are of significance to the forage and landscape grass and livestock industries. In North America, the bentgrass nematode, Anguina agrostis, reduces seed production on Agrostis tenuis and several other grass species. Anguina funesta is a seed-gall nematode that is most significant for its association with the toxigenic bacteria Rathayibacter toxicus. The wheat seed gall nematode A. tritici causes significant damage to wheat and other cereals; although it has been found in many countries worldwide, it has not been detected in the United States since 1975. Molecular methods based upon sequence variation in the ribosomal internal spacer region are useful for accurate identification of Anguina spp. Described herein are new species-specific primers and TaqMan probes for real-time polymerase chain reaction (PCR) identification of A. agrostis, A. funesta, A. tritici, and A. pacificae. Primer and probe combinations were each specific for the intended species and were sensitive enough to detect as few as 1.25 copies of nematode ribosomal DNA. PCR was also specific and sensitive in duplex assays that included genus-specific internal control primers as well as species-specific primers and probes. These standardized real-time PCR protocols should facilitate fast and accurate identification of Anguina spp. by diagnostic laboratories.

Species-specific PCR for the identification of Cooperia curticei (Nematoda: Trichostrongylidae) in sheep

2013 ◽  
Vol 88 (4) ◽  
pp. 447-452 ◽  
Author(s):  
M.R.V. Amarante ◽  
C.C. Bassetto ◽  
J.H. Neves ◽  
A.F.T. Amarante
Keyword(s):  
Geographical Location ◽  
Distinct Species ◽  
Gastrointestinal Nematodes ◽  
Internal Transcribed Spacers ◽  
Nuclear Ribosomal Dna ◽  
Oligonucleotide Primer ◽  
Specific Diagnosis ◽  
Specific Pcr ◽  
Tropical Environments ◽  
Species Specific

AbstractAgricultural ruminants usually harbour mixed infections of gastrointestinal nematodes. A specific diagnosis is important because distinct species can differ significantly in their fecundity and pathogenicity. Haemonchus spp. and Cooperia spp. are the most important gastrointestinal nematodes infecting ruminants in subtropical/tropical environments. In Brazil, C. punctata is more adapted to cattle than sheep. Additionally, C. spatulata appears to be more adapted to cattle, whereas C. curticei is more adapted to sheep. However, infection of sheep with C. punctata is common when cattle and sheep share the same pasture. Although morphological analyses have been widely used to identify nematodes, molecular methods can overcome technical limitations and help improve species-specific diagnoses. Genetic markers in the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) of nuclear ribosomal DNA (rDNA) have been used successfully to detect helminths. In the present study, the ITS-1 region was analysed and used to design a species-specific oligonucleotide primer pair to identify C. curticei. The polymerase chain reaction (PCR) product was sequenced and showed 97% similarity to C. oncophora partial ITS-1 clones and 99% similarity to the C. curticei sequence JF680982. The specificity of this primer pair was corroborated by the analysis of 17 species of helminths, including C. curticei, C. punctata and C. spatulata. Species-specific diagnosis, which has implications for rapid and reliable identification, can support studies on the biology, ecology and epidemiology of trichostrongylid nematodes in a particular geographical location.

scholarly journals Molecular Phylogenetic Analysis of Vicia L. (Fabaceae) Taxa Growing in the Southeastern Anatolia Region of Turkey: Based on Internal Transcribed Spacer (ITS)

2021 ◽  
Vol 9 (10) ◽  
pp. 1831-1839
Author(s):  
Alevcan Kaplan ◽  
Alaattin Selçuk Ertekin ◽  
Esra Gündüzler
Keyword(s):  
Phylogenetic Analysis ◽  
Internal Transcribed Spacers ◽  
Flowering Plant ◽  
Nuclear Ribosomal Dna ◽  
Molecular Phylogenetic Analysis ◽  
Conservation Strategies ◽  
Attractive Alternative ◽  
Accurate Identification ◽  
Molecular Phylogenetic ◽  
Species Specific

Leguminosae or Fabaceae is the third-largest flowering plant family and is important in terms of both food production and soil fertility. Wild Vicia species and the genetic diversity of the Southeastern Anatolia Region provide an invaluable resource for the improvement of cultivated temperate feed and legume crops. The rapid progress of technology in recent years has nowmade it possible to use modern techniques in phylogenetic studies and to examine plants in a greater detail using biochemical, cytological and molecular methods to supplement purely systematic studies. The use of molecular phylogenetic analysis is the most attractive alternative strategy for a more accurate identification of the species of the Vicia genus. In the current study, some Vicia L. taxa growing naturally in the Southeastern Anatolia Region were investigated using molecular phylogenetic analysis. Internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced in order to study the phylogenetic relationships of Vicia L. taxa. Lathyrus inconspicuous L. and Lathyrus cassius Boiss. were used as an outgroup. The ITS area was determined to be approximately 479- 672 bp. The ITS sequences were submitted to the NCBI database and accession numbers obtained. The resulting tree clearly groups and separates the sect. Narbonensis, Ervilia, Peregrinae, Lathyroides, Vicia and Cracca species but was less able to distinguish species from sect. Hypechusa and Lentopsis. The data acquired were observed to be reliable in terms of solving the taxonomical problems of the Vicia L. taxa. The morphological distinctions are greatly supported by DNA sequence studies. The species-specific markers developed in this study are useful for early detection of targeted Vicia taxa and can act as a guide to the basic data required for the evolution of systematic breeding and conservation strategies, as well as for germplasm resources.

Reverse Transcription-Quantitative real-time PCR (RT-qPCR) Assay for the Rapid Enumeration of Live Candida auris from the Healthcare Environment

2021 ◽  
Author(s):  
Bryanna Lexus Freitas ◽  
Lynn Leach ◽  
Vishnu Chaturvedi ◽  
Sudha Chaturvedi
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Ribosomal Gene ◽  
Qpcr Assay ◽  
Molecular Diagnostic ◽  
Quantitative Real Time Pcr ◽  
Candida Auris ◽  
Healthcare Environments

Ongoing healthcare-associated outbreaks of multidrug-resistant yeast Candida auris have prompted the development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcription (RT)-quantitative real-time PCR (RT-qPCR) assay to detect live C. auris in healthcare environments rapidly. This assay targeted the internal transcribed spacer 2 (ITS2) ribosomal gene by obtaining pure RNA followed by reverse transcription (ITS2 cDNA) and qPCR. ITS2 cDNA was not detectable in bleach-killed cells but detectable in heat- and ethanol-killed C. auris cells. The assay was highly sensitive, with the detection limit of ten colony-forming units (CFU) per RT-qPCR reaction. Validation studies yielded positive Ct values from sponge matrix samples spiked with 10 2 to 10 5 CFU of live C. auris while dead (bleach-killed) C. auris (10 5 /ml) or other live Candida species (10 5 /ml) had no cycle threshold (Ct) values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT-qPCR assay, confirming the concordance between culture and the PCR assay. The RT-qPCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples. Candida auris RT-qPCR assay could be an invaluable tool in surveillance efforts to control the spread of live C. auris in healthcare environments.

Rapid and accurate prediction of the frequencies of Bursaphelenchus xylophilus and B. mucronatus in mixed nematode samples using real-time species-specific PCR

Nematology ◽  
2009 ◽  
Vol 11 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Si Hyeock Lee ◽  
Il Sung Moon ◽  
Jae Soon Kang ◽  
Sang Chul Shin ◽  
Sang Gil Lee
Keyword(s):  
Real Time ◽  
Detection System ◽  
Accurate Determination ◽  
Marker Gene ◽  
Threshold Value ◽  
Nematode Species ◽  
Bursaphelenchus Xylophilus ◽  
Specific Pcr ◽  
Species Specific

AbstractAccurate detection of Bursaphelenchus xylophilus and prediction of its frequency in crude nematode samples is often hindered by the coexistence of related nematode species, such as B. mucronatus, that are morphologically similar but non-pathogenic. To establish a detection system enabling determination of the relative frequencies of B. xylophilus and B. mucronatus from field nematode samples, we developed a real-time species-specific PCR (rtssPCR) protocol which targets the substantial sequence differences in the 5S rRNA marker gene between the two nematode species. Using standard DNA mixtures of B. xylophilus and B. mucronatus in various ratios, plots of percent species proportion vs cycle threshold value (Ct value) were generated for the prediction of species frequency. The rtssPCR protocol enables the detection of target nematode frequencies as low as 0.16% at the 95% confidence level. When nematode DNA samples were extracted from the mixed specimens of B. xylophilus and B. mucronatus in various ratios and analysed by rtssPCR, the semi-log plot was nearly identical to the plot generated from standard mixed DNA samples, demonstrating that field populations of the nematodes can be directly used for rtssPCR analysis. The rapid and accurate determination of B. xylophilus or B. mucronatus frequencies by this rtssPCR protocol makes it ideal for routine monitoring and quarantine of B. xylophilus in the field.

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