Simultaneous Detection of Pathogens in Clinical Samples from Patients with Community-Acquired Pneumonia by Real-Time PCR with Pathogen-Specific Molecular Beacon Probes

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.

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A novel real-time PCR system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses

Journal of Medical Virology ◽

10.1002/jmv.21962 ◽

2010 ◽

Vol 83 (2) ◽

pp. 322-330 ◽

Cited By ~ 29

Author(s):

Harutaka Katano ◽

Motofumi Kano ◽

Tomoyuki Nakamura ◽

Takayuki Kanno ◽

Hideki Asanuma ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Clinical Samples ◽

Human Viruses

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Simultaneous Detection of Trisomies 13, 18, and 21 with Multiplex Ligation-Dependent Probe Amplification–Based Real-Time PCR

Clinical Chemistry ◽

10.1373/clinchem.2010.146472 ◽

2010 ◽

Vol 56 (9) ◽

pp. 1451-1459 ◽

Cited By ~ 13

Author(s):

Qiwei Guo ◽

Yulin Zhou ◽

Xiaobo Wang ◽

Qingge Li

Keyword(s):

Prenatal Diagnosis ◽

Real Time ◽

Real Time Pcr ◽

Karyotype Analysis ◽

Simultaneous Detection ◽

Maternal Blood ◽

Trisomy 13 ◽

Clinical Samples ◽

Dependent Probe ◽

Single Reaction

BACKGROUND Trisomies 13, 18, and 21 account for the majority of chromosomal aneuploidies detected in prenatal diagnosis. Diagnosis of these trisomies relies mainly on karyotype analysis. Several molecular methods have been developed for trisomy detection, but performance or throughput limitations of these methods currently constrain their use in routine testing. METHODS We developed multiplex ligation-dependent probe amplification–based real-time PCR (MLPA/rtPCR) to simultaneously detect these 3 trisomy conditions with a single reaction. We applied the method to DNA isolated from 144 blinded clinical samples that included 32 cases of trisomy 21, 11 cases of trisomy 18, 1 case of trisomy 13, and 100 unaffected control samples; results were compared with karyotype analysis. RESULTS As judged by the results of the karyotype analysis, MLPA/rtPCR correctly detected all 44 cases of trisomy in the analysis of the blinded clinical samples. The method was able to detect a change in chromosome dosage as low as 1.2-fold. CONCLUSIONS This novel PCR-based technology simultaneously identified 3 types of trisomy in a single reaction and accurately detected trisomy with mosaicism, while reducing assay times and costs compared with conventional methods. The MLPA/rtPCR approach may have applicability in noninvasive prenatal diagnosis with maternal blood samples.

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Development and application of two multiplex real-time PCR assays for detection and speciation of bacterial pathogens in the koala

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638718770490 ◽

2018 ◽

Vol 30 (4) ◽

pp. 523-529 ◽

Cited By ~ 6

Author(s):

Lyndal S. Hulse ◽

Danica Hickey ◽

Jessica M. Mitchell ◽

Kenneth W. Beagley ◽

William Ellis ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bacterial Pathogens ◽

Simultaneous Detection ◽

Turnaround Time ◽

Clinical Samples ◽

Phascolarctos Cinereus ◽

In The Wild ◽

Labeled Probe ◽

Free Ranging

Infectious diseases have contributed to the decline in the health of koala ( Phascolarctos cinereus) populations in the wild in some regions of Australia. Herein we report the development and validation of 2 multiplex real-time PCR (rtPCR) panels for the simultaneous detection of Mycoplasma spp., Ureaplasma spp., Bordetella bronchiseptica, and Chlamydia, including speciation and quantification of Chlamydia, in ocular, reproductive, and nasal swab samples in addition to semen and male urogenital and reproductive tissues, from koalas. Each rtPCR panel was developed for use as a single-tube reaction using pathogen-specific primers and fluorescently labeled probe sets. DNA extracted from reference strains and isolates was used for validation of sequence gene targets for the multiplex rtPCR panels. Each panel was shown to be sensitive and specific in detecting and differentiating the bacterial pathogens. The multiplex rtPCR panels were used to screen clinical samples from free-ranging and hospitalized koalas for multiple pathogens simultaneously. The multiplex rtPCR will improve turnaround time compared to individual-pathogen rtPCR methods used, to date, for confirmation of diagnosis and will provide the wildlife clinician with the ability to make treatment decisions more rapidly.

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Molecular beacon-based real-time PCR detection of primary isolates of Salmonella Typhimurium and Salmonella Enteritidis in environmental and clinical samples

BMC Microbiology ◽

10.1186/1471-2180-9-97 ◽

2009 ◽

Vol 9 (1) ◽

pp. 97 ◽

Cited By ~ 46

Author(s):

Andreas V Hadjinicolaou ◽

Victoria L Demetriou ◽

Maria A Emmanuel ◽

Charalambos K Kakoyiannis ◽

Leondios G Kostrikis

Keyword(s):

Salmonella Typhimurium ◽

Real Time ◽

Real Time Pcr ◽

Salmonella Enteritidis ◽

Molecular Beacon ◽

Pcr Detection ◽

Clinical Samples

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Molecular beacon based real-time PCR assay for simultaneous detection of Listeria monocytogenes and Salmonella spp. in dairy products

Dairy Science and Technology ◽

10.1007/s13594-011-0007-8 ◽

2011 ◽

Vol 91 (3) ◽

pp. 373-382 ◽

Cited By ~ 3

Author(s):

Jitender Singh ◽

Virender K. Batish ◽

Sunita Grover

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Dairy Products ◽

Molecular Beacon ◽

Simultaneous Detection ◽

Pcr Assay ◽

Salmonella Spp

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Assessment of real-time PCR for diagnosis ofMycoplasma pneumoniaepneumonia in pediatric patients

Canadian Journal of Microbiology ◽

10.1139/w05-118 ◽

2006 ◽

Vol 52 (2) ◽

pp. 125-129 ◽

Cited By ~ 23

Author(s):

Miyuki Morozumi ◽

Akira Ito ◽

Somay Y Murayama ◽

Keiko Hasegawa ◽

Reiko Kobayashi ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Mycoplasma Pneumoniae ◽

Real Time Pcr ◽

High Sensitivity ◽

Community Acquired Pneumonia ◽

Clinical Samples ◽

Rrna Gene ◽

The 16S Rrna Gene

We developed a real-time PCR to detect Mycoplasma pneumoniae with a primer set designed for the 16S rRNA gene. Clinical samples (n = 937) were collected from children with community-acquired pneumonia between April 2002 and March 2004 at 12 Japanese medical institutions. Sensitivity of real-time PCR was calculated as 10 colony-forming units per reaction tube using a pMP01 plasmid carrying a 225-bp target DNA fragment of the 16S rRNA gene in M. pneumoniae M129, a standard strain. Results, obtained within 2 h, were compared with those of conventional culture and serologic methods. Of all cases tested, 151 (16.4%) and 129 (13.8%) were positive for M. pneumoniae by real-time PCR and by culture, respectively. Among the 151 cases, almost all of those tested serologically by passive agglutination showed a rise in M. pneumoniae antibody titre between acute and convalescent sera. We conclude that this real-time PCR can identify M. pneumoniae rapidly and fulfills the need for rapid identification, high sensitivity, and high specificity.Key words: real-time PCR, Mycoplasma pneumoniae identification, pediatrics, community-acquired pneumonia, Mycoplasma pneumoniae culture.

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Simultaneous Detection of Seven Human Coronaviruses by Multiplex PCR and MALDI-TOF MS

COVID ◽

10.3390/covid2010002 ◽

2021 ◽

Vol 2 (1) ◽

pp. 5-17

Author(s):

Tingting Liu ◽

Lin Kang ◽

Yanwei Li ◽

Jing Huang ◽

Zishuo Guo ◽

...

Keyword(s):

Multiplex Pcr ◽

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Detection Sensitivity ◽

Clinical Samples ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Human Coronaviruses ◽

Tof Ms

Human coronaviruses (HCoVs) are associated with a range of respiratory symptoms. The discovery of severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome, and SARS-CoV-2 pose a significant threat to human health. In this study, we developed a method (HCoV-MS) that combines multiplex PCR with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), to detect and differentiate seven HCoVs simultaneously. The HCoV-MS method had high specificity and sensitivity, with a 1–5 copies/reaction detection limit. To validate the HCoV-MS method, we tested 163 clinical samples, and the results showed good concordance with real-time PCR. Additionally, the detection sensitivity of HCoV-MS and real-time PCR was comparable. The HCoV-MS method is a sensitive assay, requiring only 1 μL of a sample. Moreover, it is a high-throughput method, allowing 384 samples to be processed simultaneously in 30 min. We propose that this method be used to complement real-time PCR for large-scale screening studies.

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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.

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