Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae

Abstract This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10−6, 10−6 and 10−4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.

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Simultaneous Detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, Three Major Fish Pathogens, by Multiplex PCR

Applied and Environmental Microbiology ◽

10.1128/aem.68.10.5177-5180.2002 ◽

2002 ◽

Vol 68 (10) ◽

pp. 5177-5180 ◽

Cited By ~ 70

Author(s):

A. del Cerro ◽

I. Marquez ◽

J. A. Guijarro

Keyword(s):

Multiplex Pcr ◽

Simultaneous Detection ◽

Aeromonas Salmonicida ◽

16S Rrna Genes ◽

Rrna Genes ◽

Yersinia Ruckeri ◽

Pcr Assay ◽

Fish Pathogens ◽

Flavobacterium Psychrophilum ◽

Multiplex Pcr Assay

ABSTRACT A multiplex PCR assay based on the 16S rRNA genes was developed for the simultaneous detection of three major fish pathogens, Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri. The assay proved to be specific and as sensitive as each single PCR assay, with detection limits in the range of 6, 0.6, and 27 CFU for A. salmonicida, F. psychrophilum, and Y. ruckeri, respectively. The assay was useful for the detection of the bacteria in artificially infected fish as well as in fish farm outbreaks. Results revealed that this multiplex PCR system permits a specific, sensitive, reproducible, and rapid method for the routine laboratory diagnosis of infections produced by these three bacteria.

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MULTIPLEX POLYMERASE CHAIN REACTION ASSAY FOR THE DETECTION OF FIVE COMMON ENDOPARASITE INFESTATIONS IN LABORATORY RODENTS

Taiwan Veterinary Journal ◽

10.1142/s1682648520500110 ◽

2020 ◽

pp. 1-10

Author(s):

Chien-Hao Chen ◽

Ming-Hseng Wang ◽

Cho-Hua Wan

Keyword(s):

Multiplex Pcr ◽

Laboratory Animal ◽

Early Stage ◽

Housekeeping Gene ◽

Diagnostic Methods ◽

Rrna Genes ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Laboratory Rodents ◽

Infestation Status

Rodent pinworms, Spironucleus muris and Tritrichomonas muris are the endoparasites that should be monitored and excluded from laboratory animal colonies. Nevertheless, traditional diagnostic methods may not efficiently detect and accurately demonstrate the endoparasite infestation status. In this study, we developed a multiplex PCR assay targeting the rRNA genes to simultaneously detect and differentiate five endoparasites, including Syphacia obvelata, Syphacia muris, Aspiculuris tetraptera, Spironucleus muris, and T. muris, as well as a housekeeping gene in feces. The multiplex PCR could identify an equivalent infection of pinworm, Spironucleus muris and T. muris, with a detection limit of as few as 10 copies. Furthermore, dual infections with up to 100-fold differences and triple infections with 10-fold differences in parasite loads can also be detected. In comparison of traditional methods with the multiplex PCR assay, 76 rodents from 11 research colonies and 3 pet shops and additional 27 fecal samples from laboratory rodents were screened for the infestation status of the five endoparasites. The multiplex PCR had higher sensitivity (97.2–100%) and accuracy (99–100%) than those of the traditional antemortem (sensitivity: 83–100%; accuracy: 94–100%) and postmortem methods (sensitivity: 75–100%; accuracy: 92.1–100%). In addition, an early stage of S. obvelata contamination in a SPF laboratory animal colony was also successfully detected by this multiplex PCR assay. This Pinworm/Spironucleus/Tritrichomonas/Actin Multiplex PCR assay should be a powerful tool to screen endoparasite infestations in laboratory colonies without animal sacrifice.

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Rapid Estimation of Numbers of Fecal Bacteroidetes by Use of a Quantitative PCR Assay for 16S rRNA Genes

Applied and Environmental Microbiology ◽

10.1128/aem.70.9.5695-5697.2004 ◽

2004 ◽

Vol 70 (9) ◽

pp. 5695-5697 ◽

Cited By ~ 118

Author(s):

Linda K. Dick ◽

Katharine G. Field

Keyword(s):

16S Rrna ◽

Fecal Pollution ◽

16S Rrna Genes ◽

Rrna Genes ◽

Fecal Indicators ◽

Pcr Assay ◽

Fecal Indicator ◽

Quantification Method ◽

Quantitative Pcr Assay ◽

Nuclease Assay

ABSTRACT Assessment of health risk associated with fecal pollution requires a reliable fecal indicator and a rapid quantification method. We report the development of a Taq nuclease assay for enumeration of 16S rRNA genes of Bacteroidetes. Sensitivity and correlation with standard fecal indicators provide experimental evidence for application of the assay in monitoring fecal pollution.

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Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection ofActinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis

Journal of Clinical Microbiology ◽

10.1128/jcm.37.11.3504-3508.1999 ◽

1999 ◽

Vol 37 (11) ◽

pp. 3504-3508 ◽

Cited By ~ 96

Author(s):

Simon Dangtuan Tran ◽

Joel D. Rudney

Keyword(s):

16S Rrna ◽

Multiplex Pcr ◽

Porphyromonas Gingivalis ◽

Healthy Subjects ◽

Simultaneous Detection ◽

Oral Bacteria ◽

16S Rrna Genes ◽

Rrna Genes ◽

Subgingival Plaque ◽

Periodontal Pathogens

Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, andPorphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and fourP. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species.A. actinomycetemcomitans, B. forsythus, andP. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays.

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A multiplex PCR assay based on 16S rRNA and hly for rapid detection of L. monocytogenes in Milk

Journal of Food Measurement & Characterization ◽

10.1007/s11694-014-9176-5 ◽

2014 ◽

Vol 8 (3) ◽

pp. 155-163 ◽

Cited By ~ 3

Author(s):

Ashwani Kumar ◽

Sunita Grover ◽

Virender Kumar Batish

Keyword(s):

16S Rrna ◽

Multiplex Pcr ◽

Rapid Detection ◽

Pcr Assay ◽

Multiplex Pcr Assay

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Development of a multiplex PCR assay for detection and identificationof a chickpea phytoplasma strain utilizing 16S rRNA and imp genesspecific primers

Phytopathogenic Mollicutes ◽

10.5958/2249-4677.2019.00123.3 ◽

2019 ◽

Vol 9 (2) ◽

pp. 257

Author(s):

Madem Gurivi Reddy ◽

Govind Pratap Rao

Keyword(s):

16S Rrna ◽

Multiplex Pcr ◽

Pcr Assay ◽

Phytoplasma Strain ◽

Multiplex Pcr Assay

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Multiplex PCR for the Detection ofLactobacillus pontis and Two Related Species in a Sourdough Fermentation

Applied and Environmental Microbiology ◽

10.1128/aem.66.5.2113-2116.2000 ◽

2000 ◽

Vol 66 (5) ◽

pp. 2113-2116 ◽

Cited By ~ 21

Author(s):

Martin R. A. Müller ◽

Matthias A. Ehrmann ◽

Rudi F. Vogel

Keyword(s):

16S Rrna ◽

Multiplex Pcr ◽

Related Species ◽

Lactobacillus Reuteri ◽

Comparative Sequence Analysis ◽

Pcr Assay ◽

Variable Regions ◽

Specific Pcr ◽

Multiplex Pcr Assay ◽

Specific Pcr Assay

ABSTRACT A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis,Lactobacillus oris, and Lactobacillus reuteriand from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.

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Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium, Lactobacillus thermotolerans, in Chicken Feces

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4214-4219.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4214-4219 ◽

Cited By ~ 15

Author(s):

Abu Sadeque Md Selim ◽

Piyanuch Boonkumklao ◽

Teruo Sone ◽

Apinya Assavanig ◽

Masaru Wada ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

Pure Culture ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Pcr Assay ◽

Chicken Feces

ABSTRACT A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345T and Lactobacillus fermentum JCM 1173T, and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 × 103 cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 104 cells/g feces on day 4 and 105 cells/g feces on days 11 to 18. However, cell populations of 106 to 107 cells/g feces were detected in the second trial. The total bacterial count, measured by 4′,6-diamidino-2-phenylindole (DAPI) staining, was approximately 1011 cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.

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Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6597-6604.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6597-6604 ◽

Cited By ~ 74

Author(s):

Hebe M. Dionisi ◽

Gerda Harms ◽

Alice C. Layton ◽

Igrid R. Gregory ◽

Jack Parker ◽

...

Keyword(s):

16S Rrna ◽

Activated Sludge ◽

Real Time ◽

Real Time Pcr ◽

Power Analysis ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Pcr Assay ◽

Pcr Assays

ABSTRACT The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.

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