A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

Download Full-text

Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.00115-15 ◽

2015 ◽

Vol 53 (7) ◽

pp. 2148-2153 ◽

Cited By ~ 28

Author(s):

Xuan Qin ◽

Eileen J. Klein ◽

Emmanouil Galanakis ◽

Anita A. Thomas ◽

Jennifer R. Stapp ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Primary Cultures ◽

Clinical Samples ◽

Pcr Assay ◽

Content Type ◽

E Coli ◽

Successful Recovery

Timely accurate diagnosis of Shiga toxin-producingEscherichia coli(STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targetingstx1,stx2, andrfbEO157with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagicE. coli[EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC.E. coliO157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 usingrfbEO157, and LD-PCR results prompted successful recovery ofE. coliO157 (n= 25) and non-O157 STEC (n= 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid “mega”-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and thatE. coliO157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections.

Download Full-text

Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae

Journal of Clinical Microbiology ◽

10.1128/jcm.00493-15 ◽

2015 ◽

Vol 53 (7) ◽

pp. 2042-2048 ◽

Cited By ~ 22

Author(s):

S. W. Peterson ◽

I. Martin ◽

W. Demczuk ◽

A. Bharat ◽

L. Hoang ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Real Time ◽

Dna Sequence ◽

Real Time Pcr ◽

Nucleic Acid Amplification ◽

Clinical Samples ◽

Specific Marker ◽

Molecular Assay ◽

Pcr Assay ◽

Content Type

The incidence of antimicrobial-resistantNeisseria gonorrhoeaecontinues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) inponA,mtrR,penA,porB, and oneN. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24N. gonorrhoeae-negative NAAT specimens, and 34N. gonorrhoeae-positive NAAT specimens. Twenty-four of theN. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252N. gonorrhoeaestrains, the agreement between the DNA sequence and real-time PCR was 100% forporA,ponA, andpenA, 99.6% formtrR, and 95.2% forporB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% forporB, 95.8% forponAandmtrR, and 91.7% forpenA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins inN. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.

Download Full-text

Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

Applied and Environmental Microbiology ◽

10.1128/aem.02490-09 ◽

2010 ◽

Vol 76 (13) ◽

pp. 4387-4395 ◽

Cited By ~ 39

Author(s):

Sebastian Kirchner ◽

K. Melanie Krämer ◽

Martin Schulze ◽

Diana Pauly ◽

Daniela Jacob ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Target Genes ◽

False Negative ◽

Simultaneous Detection ◽

Locked Nucleic Acid ◽

Clinical Samples ◽

Quantitative Real Time Pcr ◽

Pcr Assay ◽

Pcr Assays

ABSTRACT Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 103 and 105 CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.

Download Full-text

Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp.

Microbial Genomics ◽

10.1099/mgen.0.000406 ◽

2020 ◽

Vol 6 (7) ◽

Author(s):

Erin P. Price ◽

Valentina Soler Arango ◽

Timothy J. Kidd ◽

Tamieka A. Fraser ◽

Thuy-Khanh Nguyen ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Type Species ◽

Simultaneous Detection ◽

Diagnostic Methods ◽

Achromobacter Xylosoxidans ◽

Resistant Bacteria ◽

Pcr Assay ◽

Content Type ◽

Link Type

Several members of the Gram-negative environmental bacterial genus Achromobacter are associated with serious infections, with Achromobacter xylosoxidans being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 Achromobacter spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus Achromobacter and for A. xylosoxidans . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans , with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive in silico analysis, and laboratory testing of 34 non- Achromobacter isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

Download Full-text

Simultaneous detection and differentiation of clinically relevant relapsing fever Borrelia with semi-multiplex real-time PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.02981-20 ◽

2021 ◽

Author(s):

Elizabeth A. Dietrich ◽

Adam J. Replogle ◽

Sarah W. Sheldon ◽

Jeannine M. Petersen

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Simultaneous Detection ◽

Clinical Samples ◽

Hard Tick ◽

Emerging Pathogens ◽

Pcr Assay ◽

Relapsing Fever ◽

Multiplex Pcr Assay

Bacterial vector-borne diseases, including Borrelia species, present a significant diagnostic, clinical, and public health challenge due to their overlapping symptoms and the breadth of causative agents and arthropod vectors. The relapsing fever (RF) borreliae encompass both established and emerging pathogens and are transmitted to humans by soft ticks, hard ticks, or lice. We developed a real-time semi-multiplex PCR assay that detects multiple RF borreliae causing human illness and classifies them into one of three groups. The groups are based on genetic similarity and include agents of soft-tick relapsing fever (B. hermsii and others), the emerging hard tick transmitted pathogen B. miyamotoi, and the agent of louse-borne relapsing fever (B. recurrentis). The real-time PCR assay uses a single primer pair designed to amplify all known pathogenic RF borreliae, and multiple TaqMan probes to allow for detection of and differentiation among the three groups. The assay detects all RF borreliae tested with an analytical limit of detection below 15 genome equivalents per reaction. Thirty isolates of RF borreliae encompassing six species were accurately identified. Thirty-nine of 41 residual specimens (EDTA whole blood, serum, or plasma) from patients with RF were detected and correctly classified. None of 42 clinical samples from patients with other infections and 46 culture specimens from non-RF bacteria were detected. The development of a single assay real-time PCR approach will help to improve diagnosis of RF by simplifying the selection of tests to aid in clinical management of acutely ill RF patients.

Download Full-text

Validation of a Multiplex Real-Time PCR Assay for Detection of Mycobacterium spp., Mycobacterium tuberculosis Complex, and Mycobacterium avium Complex Directly from Clinical Samples by Use of the BD Max Open System

Journal of Clinical Microbiology ◽

10.1128/jcm.00241-16 ◽

2016 ◽

Vol 54 (6) ◽

pp. 1644-1647 ◽

Cited By ~ 10

Author(s):

Talita T. Rocchetti ◽

Suzane Silbert ◽

Alicia Gostnell ◽

Carly Kubasek ◽

Raymond Widen

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Mycobacterium Avium ◽

Open System ◽

Real Time Pcr ◽

Clinical Samples ◽

Pcr Assay ◽

Content Type ◽

Multiplex Pcr Assay ◽

Mycobacterium Spp

A multiplex real-time PCR was validated on the BD Max open system to detect differentMycobacterium tuberculosiscomplex,Mycobacterium aviumcomplex, andMycobacteriumspp. directly from clinical samples. The PCR results were compared to those with traditional cultures. The multiplex PCR assay was found to be a specific and sensitive method for the rapid detection of mycobacteria directly from clinical specimens.

Download Full-text

A Real-Time Multiplex PCR Assay for Detection ofElizabethkingiaSpecies and Differentiation betweenElizabethkingia anophelisandE. meningoseptica

Journal of Clinical Microbiology ◽

10.1128/jcm.01619-18 ◽

2019 ◽

Vol 57 (4) ◽

Cited By ~ 3

Author(s):

Aubree J. Kelly ◽

Sandor E. Karpathy ◽

Christopher A. Gulvik ◽

Melissa L. Ivey ◽

Anne M. Whitney ◽

...

Keyword(s):

Comparative Genomics ◽

Real Time ◽

Nosocomial Infections ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Diagnostic Tools ◽

Pcr Assay ◽

Content Type ◽

Multiplex Pcr Assay ◽

Elizabethkingia Meningoseptica

ABSTRACTNosocomial infections ofElizabethkingiaspecies can have fatal outcomes if not identified and treated properly. The current diagnostic tools available require culture and isolation, which can extend the reporting time and delay treatment. Using comparative genomics, we developed an efficient multiplex real-time PCR for the simultaneous detection of all known species ofElizabethkingia, as well as differentiating the two most commonly reported species,Elizabethkingia anophelisandElizabethkingia meningoseptica.

Download Full-text

Detection of Mycobacteria, Mycobacterium avium Subspecies, and Mycobacterium tuberculosis Complex by a Novel Tetraplex Real-Time PCR Assay

Journal of Clinical Microbiology ◽

10.1128/jcm.03168-14 ◽

2015 ◽

Vol 53 (3) ◽

pp. 930-940 ◽

Cited By ~ 21

Author(s):

Iker A. Sevilla ◽

Elena Molina ◽

Natalia Elguezabal ◽

Valentín Pérez ◽

Joseba M. Garrido ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Mycobacterium Avium ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Reference Method ◽

Clinical Samples ◽

Dna Amount ◽

Content Type ◽

Specificity And Sensitivity

Mycobacterium tuberculosiscomplex,Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection ofMycobacteriumgenus,M. aviumsubspecies, andM. tuberculosiscomplex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n= 38) and nonmycobacterial (n= 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n= 57), archival clinical samples (n= 233), and strains isolated from various hosts (n= 147). The minimum detectable DNA amount per reaction was 50 fg forM. bovisBCG andM. kansasiiand 5 fg forM. aviumsubsp.hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identifyM. aviumandM. tuberculosiscomplex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.

Download Full-text

Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

Journal of Clinical Microbiology ◽

10.1128/jcm.01962-15 ◽

2015 ◽

Vol 53 (12) ◽

pp. 3935-3937 ◽

Cited By ~ 5

Author(s):

Daniel Golparian ◽

Stina Boräng ◽

Martin Sundqvist ◽

Magnus Unemo

Keyword(s):

Neisseria Gonorrhoeae ◽

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Molecular Detection ◽

Analytical Sensitivity ◽

Pcr Assay ◽

Content Type ◽

Dna Assay ◽

Supplementary Test

The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection ofNeisseria gonorrhoeae.

Download Full-text