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

2018 ◽  
Vol 30 (4) ◽  
pp. 523-529 ◽  
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.

Simultaneous Detection of Selected Fungal and Bacterial Pathogens by Real Time PCR in Patients with Fever and Neutropenia

2018 ◽  
Vol 18 ◽  
pp. S186
Author(s):  
Mervat Mansour ◽  
Iman Ragab ◽  
Marwa Saad ◽  
Eman Elkholy ◽  
Ebtsam Abdel-Hafez
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Pathogens ◽  
Simultaneous Detection

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

2020 ◽  
Vol 59 (1) ◽  
pp. e01986-20
Author(s):  
Ibne Karim M. Ali ◽  
Shantanu Roy
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Small Subunit ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Rdna Sequences ◽  
Appropriate Treatment

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.

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

2010 ◽  
Vol 83 (2) ◽  
pp. 322-330 ◽  
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

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

2010 ◽  
Vol 56 (9) ◽  
pp. 1451-1459 ◽  
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.

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

COVID ◽  
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.

scholarly journals Development of a 5′-Nuclease Real-Time PCR Assay Targeting fliP for the Rapid Identification of Burkholderia mallei in Clinical Samples

2006 ◽  
Vol 52 (2) ◽  
pp. 307-310 ◽  
Author(s):  
Herbert Tomaso ◽  
Holger C Scholz ◽  
Sascha Al Dahouk ◽  
Meike Eickhoff ◽  
Thomas M Treu ◽  
...  
Keyword(s):  
Real Time ◽  
United Arab Emirates ◽  
Real Time Pcr ◽  
Bacterial Load ◽  
Turnaround Time ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Burkholderia Mallei ◽  
Pcr Assay ◽  
Bacterial Dna

Abstract Background: Burkholderia mallei is a potential biological agent that causes glanders or farcy in solipeds, a disease notifiable to the Office International des Epizooties (OIE). The number of reported outbreaks has increased steadily during the last decade, but diagnosis is hampered by the low bacterial load in infected tissues and excretions. Methods: We developed a B. mallei-specific 5′-nuclease real-time PCR assay that targets the fliP gene of B. mallei and includes an internal amplification control. Specificity was assessed with 19 B. mallei strains, 27 Burkholderia pseudomallei strains, other Burkholderia strains of 29 species, and clinically relevant non-Burkholderia organisms. Results: Amplification products were observed in all B. mallei strains but in no other bacteria. The linear range of the B. mallei real-time PCR covered concentrations from 240 pg to 70 fg of bacterial DNA/reaction. The detection limit was 60 fg of B. mallei DNA. The clinical applicability of the assay was demonstrated by use of organ samples from diseased horses of a recent outbreak that was reported to the OIE by the United Arab Emirates in 2004. Conclusions: Compared with conventional PCR, our rapid 5′-nuclease real-time PCR assay for the specific identification of B. mallei has a lower risk of carryover contamination and eliminates the need for post-PCR manipulations. This real-time PCR assay also shortens the turnaround time for results and has the potential for automation.

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