scholarly journals Application of pbp1A PCR in Identification of Penicillin-Resistant Streptococcus pneumoniae

1999 ◽  
Vol 37 (3) ◽  
pp. 628-632 ◽  
Author(s):  
Mignon du Plessis ◽  
Anthony M. Smith ◽  
Keith P. Klugman
Keyword(s):  
Streptococcus Pneumoniae ◽  
Clinical Isolates ◽  
Penicillin Resistance ◽  
Binding Region ◽  
Pcr Assay ◽  
Specific Primers ◽  
Predictive Values ◽  
Species Specific ◽  
Seminested Pcr

A seminested PCR assay, based on the amplification of the pneumococcal pbp1A gene, was developed for the detection of penicillin resistance in clinical isolates of Streptococcus pneumoniae. The assay was able to differentiate between intermediate (MICs = 0.25 to 0.5 μg/ml) and higher-level (MICs = ≥1 μg/ml) resistance. Two species-specific primers, 1A-1 and 1A-2, which amplified a 1,043-bp region of thepbp1A penicillin-binding region, were used for pneumococcal detection. Two resistance primers, 1A-R1 and 1A-R2, were designed to bind to altered areas of the pbp1A gene which, together with the downstream primer 1A-2, amplify DNA from isolates with penicillin MICs of ≥0.25 and ≥1 μg/ml, respectively. A total of 183 clinical isolates were tested with the pbp1A assay. For 98.3% (180 of 183) of these isolates, the PCR results obtained were in agreement with the MIC data. The positive and negative predictive values of the assay were 100 and 91%, respectively, for detecting strains for which the MICs were ≥0.25 μg/ml and were both 100% for strains for which the MICs were ≥1 μg/ml.

Specific PCR Detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in Chicken Meat

2009 ◽  
Vol 72 (7) ◽  
pp. 1491-1495 ◽  
Author(s):  
DANIELA PENTIMALLI ◽  
NICOLETTE PEGELS ◽  
TERESA GARCÍA ◽  
ROSARIO MARTÍN ◽  
ISABEL GONZÁLEZ
Keyword(s):  
Pcr Amplification ◽  
Chicken Meat ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Gyra Gene ◽  
Specific Primers ◽  
Specific Pcr ◽  
Arcobacter Butzleri ◽  
Species Specific

An enrichment PCR assay using species-specific primers was developed for the detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in chicken meat. Primers for A. cryaerophilus, A. skirrowii, and A. cibarius were designed based on the gyrA gene to amplify nucleic acid fragments of 212, 257, and 145 bp, respectively. The A. butzleri–specific primers were designed flanking a 203-bp DNA fragment in the 16S rRNA gene. The specificity of the four primer pairs was assessed by PCR analysis of DNA from a panel of Arcobacter species, related Campylobacter, Helicobacter species, and other food bacteria. The applicability of the method was then validated by testing 42 fresh retail-purchased chicken samples in the PCR assay. An 18-h selective preenrichment step followed by PCR amplification with the four Arcobacter primer sets revealed the presence of Arcobacter spp. in 85.7% of the retail chicken samples analyzed. A. butzleri was the only species present in 50% of the samples, and 35.7% of the samples were positive for both A. butzleri and A. cryaerophilus. A. skirrowii and A. cibarius were not detected in any of the chicken samples analyzed. The enrichment PCR assay developed is a specific and rapid alternative for the survey of Arcobacter contamination in meat.

Development of a multiplex PCR assay for the detection of key genes associated with Pasteurella multocida subspecies

2021 ◽  
pp. 104063872110634
Author(s):  
Barbara Ujvári ◽  
Hubert Gantelet ◽  
Tibor Magyar
Keyword(s):  
Multiplex Pcr ◽  
Pasteurella Multocida ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Multiplex Pcr Assay ◽  
Subspecies Level ◽  
Species Specific ◽  
Reference Strains

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species–specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test–based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.

scholarly journals Genomic Analyses of DNA Transformation and Penicillin Resistance in Streptococcus pneumoniae Clinical Isolates

2013 ◽  
Vol 58 (3) ◽  
pp. 1397-1403 ◽  
Author(s):  
Fereshteh Fani ◽  
Philippe Leprohon ◽  
George G. Zhanel ◽  
Michel G. Bergeron ◽  
Marc Ouellette
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Proteins ◽  
Resistance Mechanisms ◽  
Clinical Isolates ◽  
Penicillin Resistance ◽  
Dna Transformation ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
Genomic Analyses ◽  
Genome Scale

ABSTRACTAlterations in penicillin-binding proteins, the target enzymes for β-lactam antibiotics, are recognized as primary penicillin resistance mechanisms inStreptococcus pneumoniae. Few studies have analyzed penicillin resistance at the genome scale, however, and we report the sequencing ofS. pneumoniaeR6 transformants generated while reconstructing the penicillin resistance phenotypes from three penicillin-resistant clinical isolates by serial genome transformation. The genome sequences of the three last-level transformants T2-18209, T5-1983, and T3-55938 revealed that 16.2 kb, 82.7kb, and 137.2 kb of their genomes had been replaced with 5, 20, and 37 recombinant sequence segments derived from their respective parental clinical isolates, documenting the extent of DNA transformation between strains. A role in penicillin resistance was confirmed for some of the mutations identified in the transformants. Several multiple recombination events were also found to have happened at single loci coding for penicillin-binding proteins (PBPs) that increase resistance. Sequencing of the transformants with MICs for penicillin similar to those of the parent clinical strains confirmed the importance of mosaic PBP2x, -2b, and -1a as a driving force in penicillin resistance. A role in resistance for mosaic PBP2a was also observed for two of the resistant clinical isolates.

scholarly journals PCR Detection of Mixed and Zoonoses Malaria Using Plasmodium spp Dynein Light Chain (dlc-tctex) Gene

2019 ◽  
pp. 1-12
Author(s):  
Maureen W. Kariuki ◽  
Elijah K. Githui ◽  
Andrew G. McArthur ◽  
Rashid A. Aman ◽  
Nyamu M. Njagi ◽  
...  
Keyword(s):  
Malaria Diagnosis ◽  
Clinical Malaria ◽  
Cross Reactivity ◽  
Pcr Detection ◽  
Clinical Samples ◽  
Dynein Light Chain ◽  
Pcr Assay ◽  
Specific Primers ◽  
Dynein Light Chains ◽  
Species Specific

Novel gene targets are needed in accurate diagnosis of malaria. Previous studies show that the dynein light chains (dlc) in Plasmodium are uniquely conserved within the species, possibly due to their role as the cargo adptor moiety. This study aimed at the development of PCR assay for the detection of Plasmodium based on the (dlc-Tctex) as a genus and species-specific tool in malaria diagnosis. Multiple primers were designed based on Plasmodium spp dlc(Tctex) genes. The primers were applied on PCR to detect malaria on clinical samples and on laboratory maintained isolates of P. falciparum and P. vivax for human infecting species and P. knowlesi and P. cynomolgi for zoonoses infection involving primates. The amplified PCR fragments were gene cleaned and sequenced. BLASTn e-values output from the raw nucleotide queries supports that the genes are uniquely conserved.  Species-specific primers amplified P.  falciparum infections with no cross-reactivity to P. vivax, P. knowlesi or P. cynomolgi species. In this assay only 11 out of the 30 microscope positive malaria positive clinical blood samples were positive for PCR detection of P. falciparum infection. Primers designed for Plasmodium genus amplified the target band in all clinical malaria samples but also had another specific band amplification. This preliminary data demonstrate that a species-specific dlc(Tctex) PCR assay can be used for detection of P. falciparum and optimized genus primers can be applied to differentiate mixed malaria infections.

scholarly journals Detection of Erysiphe necator in Air Samples Using the Polymerase Chain Reaction and Species-Specific Primers

2007 ◽  
Vol 97 (10) ◽  
pp. 1290-1297 ◽  
Author(s):  
Jennifer S. Falacy ◽  
Gary G. Grove ◽  
Walter F. Mahaffee ◽  
Heather Galloway ◽  
Dean A. Glawe ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Powdery Mildew ◽  
Bud Burst ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Primers ◽  
Erysiphe Necator ◽  
Air Samples ◽  
Polymerase Chain ◽  
Species Specific

A polymerase chain reaction (PCR) assay employing species-specific primers was developed to differentiate Erysiphe necator from other powdery mildews common in the northwest United States. DNA was extracted from mycelia, conidia, and/or chasmothecia that were collected from grape leaves with a Burkard cyclonic surface sampler. To differentiate E. necator from other erysiphaeceous fungi, primer pairs Uncin144 and Uncin511 were developed to select unique sequences of the internal transcribed spacer regions of E. necator. Using these primers in PCR amplifications, a 367-bp amplicon specific to E. necator was generated, but no amplicons were generated from other erysiphaceous species collected from 48 disparate hosts representing 26 vascular plant families. The PCR limit of detection was one to five conidia of E. necator placed directly into reaction mixtures or 100 to 250 conidia placed on glass rods coated with silicon grease. During field studies, this PCR assay facilitated the detection of E. necator inoculum in air samples within hours of sample rod collection and prior to disease onset. Amplification of E. necator DNA did not occur when the PCR assay was conducted on vineyard air samples collected while grapes were dormant or during periods when vine growth occurred but E. necator remained dormant. The initial PCR detection of E. necator of the season occurred during seasonal ascospore releases caused by precipitation events between bud burst and the prebloom period during the 3 years of the study. Detection ceased for 7 to 11 days following ascospore release and then resumed several days prior to the observance of microscopic symptoms and signs of powdery mildew in the field. Results of this study represent the initial step toward the goal of incorporating an inoculum availability component into current and future grapevine powdery mildew risk assessment models.

scholarly journals A Simple and Reliable Single Tube Septuple PCR Assay for Simultaneous Identification of Seven Meat Species

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1083
Author(s):  
Zhendong Cai ◽  
Song Zhou ◽  
Qianqian Liu ◽  
Hui Ma ◽  
Xinyi Yuan ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Animal Species ◽  
Meat Product ◽  
Meat Products ◽  
Pcr Assay ◽  
Specific Primers ◽  
Meat Species ◽  
Pcr Method ◽  
Species Specific ◽  
Simultaneous Identification

Multiplex PCR methods have been frequently used for authentication of meat product adulteration. Through screening of new species-specific primers designed based on the mitochondrial DNA sequences, a septuple PCR method is ultimately developed and optimized to simultaneously detect seven species including turkey (110 bp), goose (194 bp), pig (254 bp), sheep (329 bp), beef (473 bp), chicken (612 bp) and duck (718 bp) in one reaction. The proposed method has been validated to be specific, sensitive, robust and inexpensive. Taken together, the developed septuple PCR assay is reliable and efficient, not only to authenticate animal species in commercial meat products, but also easily feasible in a general laboratory without special infrastructures.

scholarly journals Detection and Identification of Mycobacteria by Amplification of the Internal Transcribed Spacer Regions with Genus- and Species-Specific PCR Primers

2000 ◽  
Vol 38 (11) ◽  
pp. 4080-4085 ◽  
Author(s):  
Heekyung Park ◽  
Hyunjung Jang ◽  
Cheolmin Kim ◽  
Byungseon Chung ◽  
Chulhun L. Chang ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Bacterial Species ◽  
Its Region ◽  
Pcr Primers ◽  
Culture Collection ◽  
Clinical Isolates ◽  
Mycobacterial Species ◽  
Specific Primers ◽  
Specific Pcr ◽  
Species Specific

We evaluated the usefulness of PCR assays that target the internal transcribed spacer (ITS) region for identifying mycobacteria at the species level. The conservative and species-specific ITS sequences of 33 species of mycobacteria were analyzed in a multialignment analysis. One pair of panmycobacterial primers and seven pairs of mycobacterial species-specific primers were designed. All PCRs were performed under the same conditions. The specificities of the primers were tested with type strains of 20 mycobacterial species from the American Type Culture Collection; 205 clinical isolates of mycobacteria, including 118Mycobacterium tuberculosis isolates and 87 isolates of nontuberculous mycobacteria from 10 species; and 76 clinical isolates of 28 nonmycobacterial pathogenic bacterial species. PCR with the panmycobacterial primers amplified fragments of approximately 270 to 400 bp in all mycobacteria. PCR with the M. tuberculosiscomplex-specific primers amplified an approximately 120-bp fragment only for the M. tuberculosis complex. Multiplex PCR with the panmycobacterial primers and the M. tuberculosiscomplex-specific primers amplified two fragments that were specific for all mycobacteria and the M. tuberculosis complex, respectively. PCR with M. avium complex-, M. fortuitum-, M. chelonae-, M. gordonae-, M. scrofulaceum-, andM. szulgai-specific primers amplified specific fragments only for the respective target organisms. These novel primers can be used to detect and identify mycobacteria simultaneously under the same PCR conditions. Furthermore, this protocol facilitates early and accurate diagnosis of mycobacteriosis.

Conventional PCR for molecular diagnosis of human strongyloidiasis

Parasitology ◽  
2014 ◽  
Vol 141 (5) ◽  
pp. 716-721 ◽  
Author(s):  
R. B. SITTA ◽  
F. M. MALTA ◽  
J. R. PINHO ◽  
P. P. CHIEFFI ◽  
R. C. B. GRYSCHEK ◽  
...  
Keyword(s):  
Molecular Diagnosis ◽  
Agar Plate ◽  
Strongyloides Stercoralis ◽  
Conventional Polymerase Chain Reaction ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Culture Methods ◽  
Specific Primers ◽  
Stool Samples ◽  
Species Specific

SUMMARYStrongyloidiasis is frequently asymptomatic and diagnosis of latent infection is difficult due to limitations of current parasitological and serological methods. This study aimed to verify the use of conventional polymerase chain reaction (PCR) assay for molecular diagnosis of Strongyloides stercoralis infection. Fresh stool samples were obtained from 103 individuals: 33 S. stercoralis positive, 30 positive for other parasites and 40 negative for parasitological methods. These samples were examined by the Lutz, Rugai and agar plate culture methods and conventional PCR assay. Two sets of primers (S. stercoralis species-specific and genus-specific sets), located in the 18S ribosomal RNA gene, were used for PCR. Of the 33 samples positive for S. stercoralis by parasitological methods, 28 (84·8%) were also detected by PCR assay using species-specific primers and 26 (78·8%) using genus-specific primers. Among the stool samples negative by parasitological methods, seven (17·5%) were positive by PCR using species-specific primers and two (5·0%) using genus-specific primers. In conclusion, the conventional PCR assay described in this study using a species-specific primer pair provided a molecular method for S. stercoralis diagnosis in human stool samples.

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