Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

Abstract Background Pathogens including duck-origin avian influenza virus (AIV), duck-origin Newcastle disease virus (NDV) and duck Tembusu virus (DTMUV) posed great harm to ducks and caused great economic losses to the duck industry. In this study, we aim to develop a triplex real-time polymerase chain reaction (PCR) assay to detect these three viruses as early as possible in the suspicious duck flocks. Results The detection limit of the triplex real-time PCR for AIV, NDV, and DTMUV was 1 × 101 copies/μL, which was at least 10 times higher than the conventional PCR. In addition, the triplex assay was highly specific, and won’t cross-react with other duck pathogens. Besides, the intra-day relative standard deviation and inter-day relative standard deviation were lower than 4.44% for these viruses at three different concentrations. Finally, a total of 120 clinical samples were evaluated by the triplex real-time PCR, the conventional PCR and virus isolation, and the positive rates for these three methods were 20.83, 21.67, 19.17%, respectively. Taking virus isolation as the gold standard, the diagnostic specificity and positive predictive value of the three viruses were all above 85%, while the diagnostic sensitivity and negative predictive value of the three viruses were all 100%. Conclusion The developed triplex real-time PCR is fast, specific and sensitive, and is feasible and effective for the simultaneous detection of AIV, NDV, and DTMUV in ducks.

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Comparison of Conventional PCR and Multiplex Real-Time PCR Assay for Detection of Staphylococcus aureus and Bacillus cereus in Ready-to-Eat Foods

Journal of Food Hygiene and Safety ◽

10.13103/jfhs.2021.36.2.141 ◽

2021 ◽

Vol 36 (2) ◽

pp. 141-147

Author(s):

Yu-Si Lee ◽

Seokhwan Kim ◽

Migyeong Kim ◽

Soon Han Kim

Keyword(s):

Staphylococcus Aureus ◽

Bacillus Cereus ◽

Real Time ◽

Real Time Pcr ◽

Conventional Pcr ◽

Pcr Assay

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Development of Conventional and Real-Time PCR Assays for the Rapid Detection of Group B Streptococci

Clinical Chemistry ◽

10.1093/clinchem/46.3.324 ◽

2000 ◽

Vol 46 (3) ◽

pp. 324-331 ◽

Cited By ~ 106

Author(s):

Danbing Ke ◽

Christian Ménard ◽

François J Picard ◽

Maurice Boissinot ◽

Marc Ouellette ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Culture Method ◽

Conventional Pcr ◽

Pcr Assay ◽

Group B Streptococci ◽

Standard Culture ◽

Pcr Assays ◽

Group B

Abstract Background: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity. Methods: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCyclerTM. For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays. Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only ∼30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays. Conclusion: These assays provide promising tools for the rapid detection and identification of GBS.

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Persistence of Xanthomonas campestris pv. campestris in Field Soil in Central Europe

Microorganisms ◽

10.3390/microorganisms9030591 ◽

2021 ◽

Vol 9 (3) ◽

pp. 591

Author(s):

Filip Gazdik ◽

Samuel Magnus ◽

Steven J. Roberts ◽

Rafal Baranski ◽

Jana Cechova ◽

...

Keyword(s):

Real Time ◽

Central Europe ◽

Real Time Pcr ◽

Xanthomonas Campestris ◽

Analytical Sensitivity ◽

Field Soil ◽

Conventional Pcr ◽

Pcr Assay ◽

Brassica Crop ◽

Xanthomonas Campestris Pv Campestris

Xanthomonas campestris pv. campestris (Xcc) is a bacterium that causes black rot of crucifers. The greatest losses of brassica crop production usually result from seed-borne infection, but carry-over of inoculum in field soil may also be possible. The aim of this study was to monitor persistence of Xcc in field soil in central Europe using a conventional PCR assay with hrpF primers and a two-step nested real-time PCR assay using Zur primers. The work has demonstrated that nested real-time PCR can be used to improve the analytical sensitivity for detection of Xcc in soil compared to conventional PCR, and that Xcc may persist in soil for up to two years following an infected brassica crop in central European climatic conditions.

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Evaluation of a SYBR Green Real-Time PCR Assay for Specific Detection of Pasteurella multocida in Culture and Tissue Samples from Sheep

Indian Journal of Animal Research ◽

10.18805/ijar.b-3774 ◽

2020 ◽

Author(s):

Jyoti Kumar ◽

G. G. Sonawane ◽

Fateh Singh ◽

S. Jegaveera Pandian ◽

Rajiv Kumar

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pasteurella Multocida ◽

Bacterial Species ◽

Sybr Green ◽

Economic Losses ◽

Conventional Pcr ◽

Specific Detection ◽

Pcr Assay ◽

Tissue Samples

Pasteurella multocida is one of the bacterial species involved in cases of ovine respiratory complex that has been implicated to cause significant economic losses in sheep production system worldwide. The present study was undertaken with the aim of evaluating a SYBR Green dye based real time PCR assay targeting KMT1 gene for the detection of P. multocida. The analytical specificity and sensitivity of the PCR primers were evaluated. The test showed ten-fold more sensitivity than conventional PCR and detected down to 275.5 fg/ µl of genomic DNA concentration, equivalent to 100 copies of KMT1 gene of P. multocida. The real-time PCR was found to be specific for KMT1 gene of P. multocida, as no cross reactivity was detected with a variety of known bacterial isolates. A total of 52 ovine lung tissue samples were screened for P. multocida, which showed improved level of detection as compared to conventional PCR. It is concluded that, this assay may be used as a valuable diagnostic tool for the rapid and specific detection of P. multocida. By virtue of its high throughput format and its ability to accurately identify as well as quantify the bacterial DNA, the method may be useful in large scale epidemiological studies and clarification of pathogenesis.

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Duplex PCR Methods for the Molecular Detection ofEscherichia fergusoniiIsolates from Broiler Chickens

Applied and Environmental Microbiology ◽

10.1128/aem.04169-13 ◽

2014 ◽

Vol 80 (6) ◽

pp. 1941-1948 ◽

Cited By ~ 7

Author(s):

Karen Simmons ◽

Heidi Rempel ◽

Glenn Block ◽

Vincenzo Forgetta ◽

Rolland Vaillancourt ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Broiler Chickens ◽

Hypothetical Protein ◽

Cellulose Synthase ◽

Conventional Pcr ◽

Bacterial Concentration ◽

Pcr Assay ◽

Wide Range ◽

Fraser Valley

ABSTRACTEscherichia fergusoniiis an emerging pathogen that has been isolated from a wide range of infections in animals and humans. Primers targeting specific genes, includingyliE(encoding a conserved hypothetical protein of the cellulose synthase and regulator of cellulose synthase island),EFER_1569(encoding a hypothetical protein, putative transcriptional activator for multiple antibiotic resistance), andEFER_3126(encoding a putative triphosphoribosyl-dephospho-coenzyme A [CoA]), were designed for the detection ofE. fergusoniiby conventional and real-time PCR methods. Primers were screened byin silicoPCR against 489 bacterial genomic sequences and by both PCR methods on 55 reference and field strains. Both methods were specific and sensitive forE. fergusonii, showing amplification only for this bacterium. Conventional PCR required a minimum bacterial concentration of approximately 102CFU/ml, while real-time PCR required a minimum of 0.3 pg of DNA for consistent detection. Standard curves showed an efficiency of 98.5%, with anR2value of 0.99 for the real-time PCR assay. Cecal and cloacal contents from 580 chickens were sampled from broiler farms located in the Fraser Valley (British Columbia, Canada). PresumptiveE. fergusoniiisolates were recovered by enrichment and plating on differential and selective media. Of 301 total presumptive isolates, 140 (46.5%) were identified asE. fergusoniiby biochemical profiling with the API 20E system and 268 (89.0%) using PCR methods.E. fergusoniidetection directly from cecal and cloacal samples without preenrichment was achieved with both PCR methods. Hence, the PCR methods developed in this work significantly improve the detection ofE. fergusonii.

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Optimization of duplex real-time PCR with melting-curve analysis for detecting the microsporidian parasites Nosema apis and Nosema ceranae in Apis mellifera

The Canadian Entomologist ◽

10.4039/n10-010 ◽

2010 ◽

Vol 142 (3) ◽

pp. 271-283 ◽

Cited By ~ 18

Author(s):

Karen L. Burgher-MacLellan ◽

Geoffrey R. Williams ◽

Dave Shutler ◽

Kenna MacKenzie ◽

Richard E.L. Rogers

Keyword(s):

Apis Mellifera ◽

Real Time ◽

Real Time Pcr ◽

Honey Bees ◽

Melting Curve ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Nosema Apis ◽

Conventional Pcr ◽

Pcr Assay

AbstractHoney bees, Apis mellifera (L.) (Hymenoptera: Apidae), are parasitized by the microsporidians Nosema apis (Zander) and Nosema ceranae (Fries). Molecular techniques are commonly used to differentiate between these parasites because light microscopy is inadequate. Our objectives were to (i) adapt the previously published duplex polymerase chain reaction (PCR) targeting the 16S rRNA gene of N. apis (321APIS-FOR, 321APIS-REV) and N. ceranae (218MITOC-FOR, 218MITOC-REV) using qualitative real-time PCR assay with SYBR® Green I dye (R-T PCR) and DNA melting-curve analysis, and (ii) determine whether the two Nosema species can be detected simultaneously in honey bees. Total spore counts and purified total genomic DNA were obtained from 37 bee samples (19 individual workers and 18 pooled samples of 15 workers) collected in Nova Scotia, Prince Edward Island, and Newfoundland, Canada. Overall, the prevalence of Nosema species was 86.5% (32/37 samples of bee DNA), based on conventional PCR and the optimized R-T PCR assay. The melting-curve analysis showed three groups of curve profiles that could determine the prevalence of N. apis, N. ceranae, and co-infection (21.9%, 56.2%, and 21.9%, respectively). The duplex R-T PCR assay was efficient, specific, and more sensitive than duplex conventional PCR because co-infection was identified in 5.4% (n = 2) more samples. Sequencing of R-T PCR products confirmed the results of the melting-curve analysis. Duplex R-T PCR with melting-curve analysis is a sensitive and rapid method of detecting N. apis, N. ceranae, and co-infection in honey bees.

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Molecular Detection of the Three Major Pathogenic Vibrio Species from Seafood Products and Sediments in Tunisia Using Real-Time PCR

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-205 ◽

2016 ◽

Vol 79 (12) ◽

pp. 2086-2094 ◽

Cited By ~ 4

Author(s):

MORSI GDOURA ◽

HANEN SELLAMI ◽

HANEN NASFI ◽

RAHMA TRABELSI ◽

SABEUR MANSOUR ◽

...

Keyword(s):

Human Health ◽

Real Time ◽

Real Time Pcr ◽

Ruditapes Decussatus ◽

Conventional Pcr ◽

Pcr Assay ◽

Isolation And Characterization ◽

Seafood Products ◽

Pcr Method ◽

Vibrio Spp

ABSTRACT Vibrio spp. have emerged as a serious threat to human health worldwide. V. parahaemolyticus, V. cholerae, and V. vulnificus pose a considerable public health risk in Tunisia because they cause sporadic and epidemic foodborne infections associated with the consumption of raw or undercooked contaminated seafood. More recently, toxR-positive V. alginolyticus was also reported to be a potential source of contaminated seafood. A total of 247 samples, including 113 fishes (Labrus viridis, Penaeus kerathurus, Diplodus annularis, Diplodus sparaillon, Scorparna porcus, Sarpa salpa, Dentex dentex, Scorparna scrofa, Sardinella aurita, Trachurus trachurus, Synodus saurus, Pagellus erythrinus, and Metapenaeus monoceros), 83 clams (Ruditapes decussatus species), 30 seawater samples, and 21 sediment samples were analyzed using traditional culture methods (ISO/TS 21872-1; International Organization for Standardization 2007) and a conventional PCR method for Vibrio spp. identification. A rapid, sensitive, and highly reproducible real-time PCR assay was developed to detect the three major Vibrio spp. pathogenic for humans in Tunisian seafood products and sediments. A conventional culture method found 102 (41.3%) of 247 analyzed samples positive for Vibrio spp.; a conventional PCR method found 126 (51%) of the 247 samples positive. Real-time PCR assay found 126 (51.1%) samples positive; V. alginolyticus toxR was the most common, found in 99 (78.57%) of samples, followed by V. parahaemolyticus in 26 (20.63%) and V. cholerae in 1 (0.7%). All culture-positive samples were PCR positive. However, 24 samples that were positive by conventional PCR and real-time PCR were culture negative. Our findings indicate that retail seafood is commonly contaminated with Vibrio spp. and presents a potential risk to human health in Tunisia. These data also indicate that real-time PCR can provide sensitive species-specific detection of Vibrio spp. in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.

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Early Detection of Leifsonia xyli subsp. xyli in Sugarcane Leaves by Real-Time Polymerase Chain Reaction

Plant Disease ◽

10.1094/pdis-91-4-0430 ◽

2007 ◽

Vol 91 (4) ◽

pp. 430-434 ◽

Cited By ~ 27

Author(s):

M. P. Grisham ◽

Y.-B. Pan ◽

E. P. Richard

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Close Agreement ◽

Leaf Tissue ◽

Conventional Pcr ◽

Chain Reaction ◽

Pcr Assay ◽

Polymerase Chain ◽

Pcr Assays

A real-time, polymerase chain reaction (PCR) assay was developed for detecting Leifsonia xyli subsp. xyli in sugarcane leaf tissue. Real-time PCR assays were conducted on the youngest, fully expanded leaf of three cultivars collected bi-weekly from field nurseries between 11 April and 19 July 2005. L. xyli subsp. xyli infection was detected in leaves collected at all sampling dates, including those from 1-month-old plants on 11 April. Assays conducted on older, more rapidly growing plants (28 July and 21 October 2005) indicated that leaf position affects assay efficiency. Conventional PCR was less efficient than real-time PCR for detecting L. xyli subsp. xyli in leaf tissue. Real-time PCR was used to rank cultivars for susceptibility to L. xyli subsp. xyli infection based on the relative titer of L. xyli subsp. xyli in leaves of inoculated, 3- and 4-month-old greenhouse-grown plants. The ranking of cultivars by real-time PCR was in close agreement with the ranking determined by tissue-blot enzyme immunoassay performed on tissue from 7- to 9-month-old stalks.

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Development of a Real-Time PCR Assay for Detection of Kudoa iwatai (Myxosporea: Multivalvulida) in Japanese Seabass (Lateolabrax japonicus)

Journal of Food Protection ◽

10.4315/0362-028x.jfp-18-089 ◽

2018 ◽

Vol 81 (8) ◽

pp. 1346-1350

Author(s):

NATSUKO IIDA-AOYAMA ◽

TETSUYA HARADA ◽

TAKAO KAWAI ◽

HIROSHI YOKOYAMA ◽

KENTARO KAWATSU

Keyword(s):

Real Time ◽

Real Time Pcr ◽

18S Rdna ◽

Conventional Pcr ◽

Lateolabrax Japonicus ◽

Pcr Assay ◽

Foreign Substance ◽

Highly Sensitive ◽

Fish Samples ◽

Japanese Seabass

ABSTRACT Kudoa iwatai, a myxosporean parasite, has low host fish specificity, and consumers encounter commercial marine fish or marketed marine fish infected with this parasite in Japan. Although the presence of this parasite infection in fish samples is traditionally determined by the microscopic morphological examination of extracted spores, this method lacks sensitivity and specificity. In this study, we developed a real-time PCR assay for the detection of K. iwatai 18S rDNA to achieve the rapid and specific identification of K. iwatai in foreign substance inspection. We also evaluated the usefulness of real-time PCR for Japanese seabass (Lateolabrax japonicus) with or without K. iwatai cysts. Our real-time PCR assay was able to reliably detect the target plasmid DNA over a 7-log range (from 4.0 × 101 to 4.0 × 107 copies per reaction) and displayed a linear relationship, with a correlation of determination value of 0.9993 and slope of −3.3651. Moreover, the mean value of the intra-assay coefficient of variation was 0.89% in triplicate assays, and the detection limit of this method was 2.5 copies of K. iwatai 18S rDNA per reaction. The sensitivity of the real-time PCR was the same or higher than that of an established conventional PCR when DNA extracts from eight Japanese seabass with or without K. iwatai were used as templates. The specificity of the real-time PCR was comparable with that of conventional PCR by using DNA extracts from fish samples infected with nine Kudoa species. Together, these results indicate that our real-time PCR assay is highly sensitive, reproducible, and specific for detecting K. iwatai 18S rDNA in foreign substance inspection. We believe that this highly sensitive real-time PCR may also be useful for understanding the gastrointestinal diseases associated with K. iwatai and for studying the yet unknown life cycle of K. iwatai.

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