Efficient differentiation of Corynebacterium striatum , Corynebacterium amycolatum and Corynebacterium xerosis clinical isolates by multiplex PCR using novel species-specific primers

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.

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Rapid identification of probioticLactobacillusspecies by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA

FEMS Microbiology Letters ◽

10.1016/j.femsle.2004.08.049 ◽

2004 ◽

Vol 239 (2) ◽

pp. 267-275 ◽

Cited By ~ 62

Author(s):

Hyuk-Sang Kwon ◽

Eun-Hee Yang ◽

Seung-Woo Yeon ◽

Byoung-Hwa Kang ◽

Tae-Yong Kim

Keyword(s):

16S Rrna ◽

Multiplex Pcr ◽

Rapid Identification ◽

23S Rrna ◽

Specific Primers ◽

Species Specific

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Development of novel species-specific primers for species identification of the Lactobacillus casei group based on RAPD fingerprints

Journal of the Science of Food and Agriculture ◽

10.1002/jsfa.3658 ◽

2009 ◽

Vol 89 (11) ◽

pp. 1831-1837 ◽

Cited By ~ 19

Author(s):

Chien-Hsun Huang ◽

Fwu-Ling Lee

Keyword(s):

Species Identification ◽

Lactobacillus Casei ◽

Novel Species ◽

Specific Primers ◽

Rapd Fingerprints ◽

Species Specific ◽

Lactobacillus Casei Group

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

Journal of Clinical Microbiology ◽

10.1128/jcm.37.3.628-632.1999 ◽

1999 ◽

Vol 37 (3) ◽

pp. 628-632 ◽

Cited By ~ 17

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.

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

Journal of Veterinary Diagnostic Investigation ◽

10.1177/10406387211063438 ◽

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.

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Biotyping and Antimicrobial Susceptibility of Enterococcus faecalis and E. faecium Isolated from Urine and Stool Samples

Jundishapur Journal of Microbiology ◽

10.5812/jjm.105136 ◽

2021 ◽

Vol 13 (10) ◽

Author(s):

Gülşah Tollu ◽

Ismail Hakkı Ekin

Keyword(s):

Multiplex Pcr ◽

Antimicrobial Susceptibility ◽

Animal Health ◽

Molecular Techniques ◽

Penicillin G ◽

Clinical Approach ◽

Biochemical Tests ◽

Specific Primers ◽

Stool Samples ◽

Species Specific

Background: Enterococci are one of the opportunistic pathogenic microorganisms that can cause significant problems for human and animal health. Enterococcus faecium seems to be more resistant to antibiotics than E. faecalis. It is thought that pathogenic E. faecium can develop antibiotic resistance very quickly, and the ability to transfer this feature is considered to be an important health risk. Objectives: This study aimed to determine the prevalence, biotypes, and in vitro antimicrobial susceptibility of E. faecalis and E. faecium strains isolated from 267 routine urine and stool samples that were brought to the microbiology laboratory of Regional Training and Research Hospital of Van, with permission of the patients. Methods: In the present study, enterococci using species-specific primers to examine E. faecalis and E. faecium multiplex PCR technique was applied. Biotyping of the isolates was used to identify them as E. faecalis and E. faecium by molecular techniques, and antibiotic susceptibility of all samples was examined, as well. Results: The isolates were identified by multiplex PCR using species-specific primers for E. faecalis and E. faecium. Biotyping based on 13 biochemical tests showed that 72.5%, 12.5%, and 15% of E. faecalis strains were of biotypes I, II, and III, respectively, whereas E. faecium strains could be divided into biotype I (10%), biotype II (12.5%), biotype III (27.5%), and biotype IV (50%). Additionally, all E. faecalis strains were found to be susceptible to penicillin G and imipenem. On the other hand, 95% of the E. faecalis strains were found to be resistant to clindamycin, 77.5% to tetracycline and trimethoprim/sulfamethoxazole, 42.5% to erythromycin, 32.5% to gentamicin, and 17.5% to ciprofloxacin. Of E. faecium strains, 37.5% were found to be resistant to clindamycin, 32.5% to penicillin G, 27.5% to erythromycin and imipenem, 20% to ciprofloxacin, 17.5% to tetracycline and trimethoprim/sulfamethoxazole, 15% to gentamicin, and 5% to vancomycin. Conclusions: In conclusion, the identification of E. faecalis and E. faecium strains by PCR is reliable and faster than biochemical tests. Additionally, the results of antimicrobial susceptibility tests may provide important contributions to the clinical approach.

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Agronomically important thrips: development of species-specific primers in multiplex PCR and microarray assay using internal transcribed spacer 1 (ITS1) sequences for identification

Bulletin of Entomological Research ◽

10.1017/s000748531400073x ◽

2014 ◽

Vol 105 (1) ◽

pp. 52-59 ◽

Cited By ~ 11

Author(s):

W.B. Yeh ◽

M.J. Tseng ◽

N.T. Chang ◽

S.Y. Wu ◽

Y.S. Tsai

Keyword(s):

Multiplex Pcr ◽

Species Identification ◽

Internal Transcribed Spacer ◽

High Throughput Screening ◽

Virus Transmission ◽

Specific Primers ◽

Its1 Sequence ◽

Microarray Assay ◽

Thrips Species ◽

Species Specific

AbstractThrips, the sole vector of plantTospovirus, are major pests of many agricultural crops throughout the world. Molecular approaches have been applied in recent decades to identify these minute and morphologically difficult to distinguish insects. In this study, sequences of internal transcribed spacer 1 (ITS1) region of 15 agronomically important thrips, including several virus transmission species, have been analyzed in order to design species-specific primers for multiplex PCR and probes for microarray assay. That the ITS1 sequence distances within species were smaller than those among species suggests that the ITS1 fragment can be used for thrips species identification. The specificity and stability of these primers, combined with universal paired primers, were tested and verified in multiplex PCR. Using these specific primers as probes, microarray assay showed that PCR products of all thrips species hybridized consistently to their corresponding probes, though some signals were weak. We have demonstrated that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable, and cost-effective diagnostic tool for thrips species identification. Moreover, the DNA microarray assay is expected to extend into a reliable high-throughput screening tool for the vast numbers of thrips.

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