Development of multiplex PCR for species-specific identification of the Poaceae family based on chloroplast gene, rpoC2

Objective: Nontuberculous mycobacteria (NTM) often cause disease that is clinically indistinguishable from tuberculosis. Specific identification is important as treatment varies according to Mycobacterium species causing the infection. This study used multiplex PCR (mPCR) assay for rapid differentiation of mycobacterial growth indicator tube 960 system (MGIT) cultures as Mycobacterium tuberculosis (MTB) or NTM together with INNO LiPA Mycobacteria v2 assay (LiPA) and/or PCR sequencing of rDNA for species-specific identification of selected MTB and all NTM isolates in Kuwait. Materials and Methods: DNA was extracted from MGIT cultures (n = 1,033) grown from 664 pulmonary and 369 extrapulmonary specimens from 1,033 suspected tuberculosis patients. mPCR was performed to differentiate MTB from NTM. LiPA was performed and results were interpreted according to kit instructions. rDNA was amplified and sequenced by using panmycobacterial primers. Results: mPCR identified 979 isolates as MTB, 53 as NTM and 1 isolate as mixed culture. LiPA and/or PCR sequencing confirmed 112 of 979 selected isolates as MTB. Mixed culture contained M. tuberculosis and M. fortuitum. LiPA yielded 12 patterns and identified 10 species/species complexes among 47 NTM, M. kansasii + M. scrofulaceum in one culture and 5 isolates only at genus level. PCR sequencing yielded more specific identification for 22 isolates at the species/subspecies level. Conclusions: mPCR rapidly differentiated MTB from NTM. LiPA identified 44 of 52 NTM isolates at the species/species complex level and 2 mixed cultures. PCR sequencing yielded more specific identification at the species/subspecies level. Rapid differentiation as MTB or NTM by mPCR, followed by species-specific NTM identification by LiPA/PCR sequencing is suitable for the proper management of mycobacterial infections in Kuwait.

Download Full-text

Identification of Adult Fasciola spp. Using Matrix-Assisted Laser/Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry

Microorganisms ◽

10.3390/microorganisms9010082 ◽

2020 ◽

Vol 9 (1) ◽

pp. 82

Author(s):

Issa Sy ◽

Lena Margardt ◽

Emmanuel O. Ngbede ◽

Mohammed I. Adah ◽

Saheed T. Yusuf ◽

...

Keyword(s):

Laser Desorption ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Specific Identification ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Species Specific ◽

Tof Ms ◽

Matrix Assisted Laser ◽

Matrix Assisted Laser Desorption

Fascioliasis is a neglected trematode infection caused by Fasciola gigantica and Fasciola hepatica. Routine diagnosis of fascioliasis relies on macroscopic identification of adult worms in liver tissue of slaughtered animals, and microscopic detection of eggs in fecal samples of animals and humans. However, the diagnostic accuracy of morphological techniques and stool microscopy is low. Molecular diagnostics (e.g., polymerase chain reaction (PCR)) are more reliable, but these techniques are not routinely available in clinical microbiology laboratories. Matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a widely-used technique for identification of bacteria and fungi; yet, standardized protocols and databases for parasite detection need to be developed. The purpose of this study was to develop and validate an in-house database for Fasciola species-specific identification. To achieve this goal, the posterior parts of seven adult F. gigantica and one adult F. hepatica were processed and subjected to MALDI-TOF MS to create main spectra profiles (MSPs). Repeatability and reproducibility tests were performed to develop the database. A principal component analysis revealed significant differences between the spectra of F. gigantica and F. hepatica. Subsequently, 78 Fasciola samples were analyzed by MALDI-TOF MS using the previously developed database, out of which 98.7% (n = 74) and 100% (n = 3) were correctly identified as F. gigantica and F. hepatica, respectively. Log score values ranged between 1.73 and 2.23, thus indicating a reliable identification. We conclude that MALDI-TOF MS can provide species-specific identification of medically relevant liver flukes.

Download Full-text

Species-specific oligonucleotides and multiplex PCR for forensic discrimination of two species of scallops, Placopecten magellanicus and Chlamys islandica

Forensic Science International ◽

10.1016/j.forsciint.2006.05.043 ◽

2007 ◽

Vol 167 (1) ◽

pp. 1-7 ◽

Cited By ~ 12

Author(s):

H.D. Marshall ◽

K.A. Johnstone ◽

S.M. Carr

Keyword(s):

Multiplex Pcr ◽

Placopecten Magellanicus ◽

Chlamys Islandica ◽

Species Specific

Download Full-text

PCR-RFLP assays for species-specific identification of fungi belonging to Scopulariopsis and related genera

Medical Mycology ◽

10.1093/mmy/myy106 ◽

2018 ◽

Vol 57 (5) ◽

pp. 643-648

Author(s):

Milena Kordalewska ◽

Joanna Kalita ◽

Zofia Bakuła ◽

Anna Brillowska-Dąbrowska ◽

Tomasz Jagielski

Keyword(s):

Specific Identification ◽

Pcr Rflp ◽

Identification Of Fungi ◽

Species Specific

Download Full-text

Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.3171-3175.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 3171-3175 ◽

Cited By ~ 56

Author(s):

X. Bonjoch ◽

E. Ballesté ◽

A. R. Blanch

Keyword(s):

16S Rrna ◽

Multiplex Pcr ◽

Fecal Pollution ◽

Sensitivity Threshold ◽

Rrna Gene ◽

Specific Primers ◽

Animal Wastewater ◽

Wastewater Samples ◽

Species Specific ◽

Different Origins

ABSTRACT Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

Download Full-text

Rapid Species Identification of Cooked Poisonous Mushrooms by Using Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.02082-07 ◽

2008 ◽

Vol 74 (10) ◽

pp. 3306-3309 ◽

Cited By ~ 24

Author(s):

Kazuhiko Maeta ◽

Tomoya Ochi ◽

Keisuke Tokimoto ◽

Norihiro Shimomura ◽

Nitaro Maekawa ◽

...

Keyword(s):

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

Specific Test ◽

Specific Identification ◽

Specific Fluorescence ◽

Species Specific ◽

Poisonous Mushrooms

ABSTRACT Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h.

Download Full-text