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.

Effects of inactivation methods on the analysis ofBacillus atrophaeusendospores using real-time PCR and MALDI-TOF-MS

2010 ◽  
pp. NA-NA ◽  
Author(s):  
Steven R. Talbot ◽  
Heiko Russmann ◽  
Stefan Köhne ◽  
Bärbel Niederwöhrmeier ◽  
Gudrun Grote ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

scholarly journals Investigation of Fluconazole Susceptibility to Candida Albicans by MALDI-TOF MS and Real-Time PCR for CDR1, CDR2, MDR1 and ERG11

2021 ◽  
Author(s):  
Chanika Maenchantrarath ◽  
Pradchama Khumdee ◽  
Seksun Samosornsuk ◽  
Narissara Mungkornkaew ◽  
Worada Samosornsuk
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Spectral Intensity ◽  
Spontaneous Mutant ◽  
Major Peak ◽  
Maldi Tof Ms ◽  
Clinical Strains ◽  
Mass Spectral ◽  
Maldi Tof ◽  
Tof Ms

Abstract Background C. albicans is the most important yeast that caused the infection in humans; the trend of resistance to fluconazole (FLC) was also increased, while the FLC susceptibility by conventional method takes time causing the treatment failure. To investigate FLC susceptibility to C. albicans using MALDI-TOF MS and Real-time PCR for CDR1, CDR2, MDR1 and ERG11, overall, 32 C. albicans strains included 4 reference strains (3 FLC susceptible (S) and 1 FLC resistant (R), 1 spontaneous mutant strain (FLC susceptible-dose dependent, SDD) and 27 clinical strains obtained from 2 Thai University Hospitals were performed FLC susceptibility testing by Sensititre YeastOne and broth microdilution method, FLC resistant expression mechanism by Real-time PCR and the major peak determination by MALDI-TOF MS.Results The change of CDR1 and CDR2 mRNA expression were only significantly observed in SDD and R strains. Using MALDI-TOF MS, the change of mass spectral intensity at range 3376-3382 m/z (major peak) was significantly related to FLC susceptibility as SDD (decreased at 4 µg/ml and increase at 8 µg/ml), S (all increased), and R (all slightly decreased or no change) after incubation for 6 h. All 27 clinical strains showed FLC MIC susceptible (MIC range 0.25-2 µg/ml), no change in CDR1 and CDR2 expression and S major peak type. The FLC resistance C. albicans with CDR1 and CDR2 expression may possibly effect the change of mass spectral intensity at range 3376-3382 m/z. Conclusions The MALDI-TOF MS may be used to simultaneously classify and predict FLC resistant C. albicans strains associated with CDR1 and CDR2 expression. Further studies are essential to clarify the methodology and improve the reliability of this assay for routine diagnosis.

scholarly journals A dual approach employing MALDI-TOF MS and real-time PCR for fast species identification within the Enterobacter cloacae complex

2012 ◽  
Vol 328 (1) ◽  
pp. 46-53 ◽  
Author(s):  
Melanie Pavlovic ◽  
Regina Konrad ◽  
Azuka N. Iwobi ◽  
Andreas Sing ◽  
Ulrich Busch ◽  
...  
Keyword(s):  
Real Time ◽  
Species Identification ◽  
Real Time Pcr ◽  
Enterobacter Cloacae ◽  
Dual Approach ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Enterobacter Cloacae Complex ◽  
Tof Ms

scholarly journals Rapid Identification of Mycoplasma bovis Strains from Bovine Bronchoalveolar Lavage Fluid with Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry after Enrichment Procedure

2020 ◽  
Vol 58 (6) ◽  
Author(s):  
Jade Bokma ◽  
Laura Van Driessche ◽  
Piet Deprez ◽  
Freddy Haesebrouck ◽  
Marianne Vahl ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lavage Fluid ◽  
Rapid Identification ◽  
Ionization Time ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Tof Ms

ABSTRACT Mycoplasma bovis is a leading cause of pneumonia in modern calf rearing. Fast identification is essential to ensure appropriate antimicrobial therapy. Therefore, the objective of this study was to develop a protocol to identify M. bovis from bronchoalveolar lavage fluid (BALf) with matrix-assisted laser desorption ionization–time of flight mass spectrometry MALDI-TOF MS and to determine the diagnostic accuracy in comparison with other techniques. BALf was obtained from 104 cattle, and the presence of M. bovis was determined in the following three ways: (i) rapid identification of M. bovis with MALDI-TOF MS (RIMM) (BALf was enriched and after 24, 48, and 72 h of incubation and was analyzed using MALDI-TOF MS), (ii) triplex real-time PCR for M. bovis, Mycoplasma bovirhinis, and Mycoplasma dispar, and (iii) 10-day incubation on selective-indicative agar. The diagnostic accuracy of the three tests was determined with Bayesian latent class modeling (BLCM). After 24 h of enrichment, M. bovis was identified with MALDI-TOF MS in 3 out of 104 BALf samples. After 48 and 72 h of enrichment, 32/104 and 38/100 samples, respectively, were M. bovis positive. Lipase-positive Mycoplasma-like colonies were seen in 28 of 104 samples. Real-time PCR resulted in 28/104 positive and 12/104 doubtful results for M. bovis. The BLCM showed a sensitivity (Se) and specificity (Sp) of 86.6% (95% credible interval [CI], 69.4% to 97.6%) and 86.4% (CI, 76.1 to 93.8) for RIMM. For real-time PCR, Se was 94.8% (CI, 89.9 to 97.9) and Sp was 88.9% (CI, 78.0 to 97.4). For selective-indicative agar, Se and Sp were 70.5% (CI, 52.1 to 87.1) and 93.9% (CI, 85.9 to 98.4), respectively. These results suggest that rapid identification of M. bovis with MALDI-TOF MS after an enrichment procedure is a promising test for routine diagnostics in veterinary laboratories.

scholarly journals Effect of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) Alone versus MALDI-TOF MS Combined with Real-Time Antimicrobial Stewardship Interventions on Time to Optimal Antimicrobial Therapy in Patients with Positive Blood Cultures

2017 ◽  
Vol 55 (5) ◽  
pp. 1437-1445 ◽  
Author(s):  
Maya Beganovic ◽  
Michael Costello ◽  
Sarah M. Wieczorkiewicz
Keyword(s):  
Real Time ◽  
Laser Desorption ◽  
Blood Cultures ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Tof Ms ◽  
Matrix Assisted Laser

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) decreases the time to organism identification and improves clinical and financial outcomes. The purpose of this study was to evaluate the impact of MALDI-TOF MS alone versus MALDI-TOF MS combined with real-time, pharmacist-driven, antimicrobial stewardship (AMS) intervention on patient outcomes. This single-center, pre-post, quasiexperimental study evaluated hospitalized patients with positive blood cultures identified via MALDI-TOF MS combined with prospective AMS intervention compared to a control cohort with MALDI-TOF MS identification without AMS intervention. AMS intervention included: real-time MALDI-TOF MS pharmacist notification and prospective AMS provider feedback. The primary outcome was the time to optimal therapy (TTOT). A total of 252 blood cultures, 126 in each group, were included in the final analysis. MALDI-TOF MS plus AMS intervention significantly reduced the overall TTOT (75.17 versus 43.06 h; P < 0.001), the Gram-positive contaminant TTOT (48.21 versus 11.75 h; P < 0.001), the Gram-negative infection (GNI) TTOT (71.83 versus 35.98 h; P < 0.001), and the overall hospital length of stay (LOS; 15.03 versus 9.02 days; P = 0.021). The TTOT for Gram-positive infection (GPI) was improved (64.04 versus 41.61 h; P = 0.082). For GPI, the hospital LOS (14.64 versus 10.31 days; P = 0.002) and length of antimicrobial therapy 24.30 versus 18.97 days; P = 0.018) were reduced. For GNI, the time to microbiologic clearance (51.13 versus 34.51 h; P < 0.001), the hospital LOS (15.40 versus 7.90 days; P = 0.027), and the intensive care unit LOS (5.55 versus 1.19 days; P = 0.035) were reduced. To achieve optimal outcomes, rapid identification with MALDI-TOF MS combined with real-time AMS intervention is more impactful than MALDI-TOF MS alone.

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 Development of Conventional Multiplex PCR: A Rapid Technique for Simultaneous Detection of Soil-Transmitted Helminths

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 152 ◽  
Author(s):  
Vivornpun Sanprasert ◽  
Ruthairat Kerdkaew ◽  
Siriporn Srirungruang ◽  
Sarit Charuchaibovorn ◽  
Kobpat Phadungsaksawasdi ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Real Time Pcr ◽  
Intestinal Parasites ◽  
Simultaneous Detection ◽  
Diagnostic Tools ◽  
Multiple Infections ◽  
Parasitic Infections ◽  
Soil Transmitted Helminths ◽  
Stool Samples

Soil-transmitted helminths (STHs) are the most common intestinal parasites infecting humans worldwide. STH infections are a major cause of morbidity and disability. Accurate diagnostic tools are pivotal for assessing the exact prevalence of parasitic infections. Microscopic examination and culture techniques have been used to observe the presence of eggs or larvae of parasites in stool samples, but they are time-consuming and have low sensitivity. Therefore, accurate, simple, and inexpensive diagnostic techniques are still required for simultaneous detection of STH infections. Although molecular-based techniques, such as real-time PCR and multiplex real-time PCR, have been developed, they are not suitable for routine diagnosis due to the requirement for expensive reagents and instruments. In this study, we established a conventional multiplex PCR for simultaneous rapid detection of Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis in stool samples. Our results show that the multiplex PCR could detect the DNA of STHs at a very low target gene concentrations (lower than 1 pg) with no cross-amplification. Multiplex PCR had five times higher sensitivity than the formalin–ethyl acetate concentration technique (FECT) in the detection of multiple infections, and two times higher for detection of S. stercoralis. However, multiplex PCR was comparable to FECT in the detection of A. lumbricoides and N. americanus. In conclusion, this method could be used as an alternative method for the detection of STHs, especially for S. stercoralis.

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