A new molecular approach to help conclude drowning as a cause of death: Simultaneous detection of eight bacterioplankton species using real-time PCR assays with TaqMan probes

AbstractMultiplex real-time PCR assays were developed to quantify multiple species of Meloidogyne incognita, Pratylenchus penetrans, Globodera rostochiensis and Heterodera glycines in soil. The probes specific for P. penetrans and H. glycines are labelled with a fluorescence molecule, FAM, and those for M. incognita and G. rostochiensis with ROX. The primers and probes are species-specific to P. penetrans, but group-specific to the other species. DNA was extracted from suspensions containing each nematode and multiplex Cycleave® PCR assays were done for pairs of P. penetrans and M. incognita, P. penetrans and G. rostochiensis, or G. rostochiensis and H. glycines. The results revealed that the target nematode, except for H. glycines, was quantified in the presence of less than 100 times that of the other nematode (competitor), but underestimated in the presence of 1000 times the competitor. Such underestimation was solved by the use of SYBR Green I real time PCR assays targeting a single species. Multiplex PCR assay for P. penetrans and M. incognita was done using environmental DNA (eDNA) extracted from a soil naturally infested with the nematodes. Results quantified both species. Multiplex assay using eDNA may enable a sensitive and simultaneous detection of P. penetrans and M. incognita or P. penetrans and G. rostochiensis in soil although caution is needed in case the existing ratio is biased to one of the species.

Download Full-text

Development of real-time PCR assays for single and simultaneous detection of infectious bursal disease virus and chicken anemia virus

Molecular and Cellular Probes ◽

10.1016/j.mcp.2018.11.004 ◽

2019 ◽

Vol 43 ◽

pp. 58-63 ◽

Cited By ~ 3

Author(s):

Claudia Techera ◽

Gonzalo Tomás ◽

Yanina Panzera ◽

Alejandro Banda ◽

Paula Perbolianachis ◽

...

Keyword(s):

Real Time ◽

Disease Virus ◽

Real Time Pcr ◽

Infectious Bursal Disease Virus ◽

Simultaneous Detection ◽

Infectious Bursal Disease ◽

Chicken Anemia Virus ◽

Bursal Disease ◽

Pcr Assays ◽

Anemia Virus

Download Full-text

A novel real-time PCR assay panel for detection of common respiratory pathogens in a convenient, strip-tube array format

10.1101/455568 ◽

2018 ◽

Author(s):

Mohammad Rubayet Hasan ◽

Hassan Al Mana ◽

Virginia Young ◽

Patrick Tang ◽

Eva Thomas ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pathogen Detection ◽

Respiratory Infections ◽

Simultaneous Detection ◽

Respiratory Pathogens ◽

Routine Manner ◽

Pcr Assays ◽

Sensitivity Specificity ◽

Array Format

AbstractCommercial multiplex assays, built on different chemistries and platforms are widely available for simultaneous detection of pathogens that cause respiratory infections. However, these tests are often difficult to implement in a resource limited setting because of high cost. In this study, we developed and validated a method for simultaneous testing of common respiratory pathogens (Respanel) by real-time PCR in a convenient, strip-tube array format. Primers and probes for sixteen PCR assays were selected from the literature or newly designed. Following optimization of individual PCR assays, strip-tube arrays were prepared by dispensing primer-probe mixes (PPM) into two sets of 8-tube strips. Nucleic acid extracts from specimens were mixed with PCR master mix, and dispensed column-wise into 2X8-wells of a 96-well plate. PPMs from strip-tubes were then added to the wells using a multichannel pipette for real-time PCR. Individual PCR assays were optimized using previously known specimens (n=397) with 91%-100% concordance with culture, DFA or PCR results. Respanel was then tested in a routine manner at two different sites using specimens (n=147) previously tested by Qiagen Resplex I&II or Fast-Track Diagnostics Respiratory Pathogens 21 assays. The sensitivity, specificity and accuracy of Respanel were 94%, 95% and 95%, respectively, against Resplex and 88%, 100% and 99%, respectively, against FTDRP21. Respanel detected 48% more pathogens (p<0.05) than Resplex but the rate of pathogen detection was not significantly different from FTDRP21. Respanel is a convenient and inexpensive assay that is more sensitive than Resplex and comparable to FTDRP21 for the detection of common respiratory pathogens.

Download Full-text

Development of Real-Time PCR Assays for the Quantitative Detection of Epstein-Barr Virus and Cytomegalovirus, Comparison of TaqMan Probes, and Molecular Beacons

Journal of Molecular Diagnostics ◽

10.1016/s1525-1578(10)60446-1 ◽

2003 ◽

Vol 5 (1) ◽

pp. 15-20 ◽

Cited By ~ 34

Author(s):

Jiska Jebbink ◽

Xin Bai ◽

Beverly Barton Rogers ◽

D. Brian Dawson ◽

Richard H. Scheuermann ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Epstein Barr Virus ◽

Molecular Beacons ◽

Quantitative Detection ◽

Barr Virus ◽

Taqman Probes ◽

Pcr Assays ◽

Epstein Barr

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