Different Real-Time PCR Formats Compared for the Quantitative Detection of Human Cytomegalovirus DNA

Abstract Background: The aim of this study was to compare the ABI PRISM 7700 Sequence Detection System and the LightCycler to develop a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA suitable for routine hospital application. Methods: We used one exonuclease probe and five different hybridization probe sets as sequence-specific fluorescence detection formats. For the exonuclease assay and two hybridization probe sets, reproducibility and the detection limit were determined. To keep the total assay time to a minimum, we gradually shortened individual reaction steps on both instruments. Results: The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The shortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is used, the exonuclease probe can be replaced by a set of hybridization probes. All assays presented here detected HCMV DNA in a linear range from 101 to 107 HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation. Conclusions: The ABI PRISM 7700 Sequence Detection System as well as the LightCycler are useful instruments for rapid and precise online PCR detection. Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy.

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Development of Multitarget Real-Time PCR for the Rapid, Specific, and Sensitive Detection of Yersinia pestis in Milk and Ground Beef

Journal of Food Protection ◽

10.4315/0362-028x-73.1.18 ◽

2010 ◽

Vol 73 (1) ◽

pp. 18-25 ◽

Cited By ~ 19

Author(s):

KINGSLEY K. AMOAKO ◽

NORIKO GOJI ◽

TREVOR MACMILLAN ◽

KAMAL B. SAID ◽

SUSAN DRUHAN ◽

...

Keyword(s):

Detection Limit ◽

Yersinia Pestis ◽

Real Time ◽

Real Time Pcr ◽

Bacterial Species ◽

Ground Beef ◽

Quantitative Detection ◽

Virulence Plasmid ◽

Gene Target ◽

Hybridization Probes

Real-time PCR has been used previously to detect Yersinia pestis; this study applies this rapid, specific, and sensitive nucleic acid–based method to the detection and quantitation of Y. pestis specifically in food. Five sets of primers and corresponding TaqMan dual-labelled fluorogenic hybridization probes for Y. pestis were designed and optimized for specificity testing using genomic DNA from 71 bacterial strains. Four Y. pestis–specific primer and probe sets were developed, based on the virulence plasmid targets, and used to distinguish this bacterium from the various Yersinia and other bacterial species tested. An additional primer and probe set, based on a chromosomal gene target, distinguished Y. pestis and Yersinia pseudotuberculosis from the various Yersinia and other bacterial species tested. With optimized conditions, the quantitative detection limit of the probes for Y. pestis pure cultures ranged from 13 to 220 CFU. Standard curves were generated for the probes and used to determine the amplification efficiencies. The primers and probes demonstrated high amplification efficiencies, and their performance was evaluated using spiked milk and ground beef samples. The quantitative detection limit was 101 to 103 CFU/ml in milk and 102 to 105 CFU/g in ground beef without any preenrichment step. Testing the hybridization probes on food samples demonstrated the detection of Y. pestis in a foodborne application; this is the first such report, to our knowledge.

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Quantitative Detection of Trichloroethylene-Degrading Mycobacterium sp. TA27 with a Real-time PCR Product Detection System.

Microbes and Environments ◽

10.1264/jsme2.2001.109 ◽

2001 ◽

Vol 16 (2) ◽

pp. 109-116 ◽

Cited By ~ 1

Author(s):

Akiko Hashimoto ◽

Kazuhiro Iwasaki ◽

Mutsuyasu Nakajima ◽

Osami Yagi

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Detection System ◽

Quantitative Detection ◽

Pcr Product

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Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 by hybridization probe based real-time PCR

Microbiological Research ◽

10.1016/j.micres.2007.03.002 ◽

2009 ◽

Vol 164 (4) ◽

pp. 486-492 ◽

Cited By ~ 7

Author(s):

Seksan Phrommanich ◽

Sudarat Suanjit ◽

Suchart Upatham ◽

Suksiri Vichasri Grams ◽

Maleeya Kruatrachue ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Quantitative Detection ◽

Hybridization Probe ◽

Degrading Bacterium ◽

Acinetobacter Sp

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Real-Time PCR Detection of Brucella abortus: a Comparative Study of SYBR Green I, 5′-Exonuclease, and Hybridization Probe Assays

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4753-4759.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4753-4759 ◽

Cited By ~ 56

Author(s):

D. T. Newby ◽

T. L. Hadfield ◽

F. F. Roberto

Keyword(s):

Real Time ◽

Brucella Abortus ◽

Real Time Pcr ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Sybr Green ◽

Sybr Green I ◽

Fluorescence Signal ◽

Insertion Element ◽

Hybridization Probe

ABSTRACT Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5′-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.

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Real-Time PCR for Quantitative Detection of Bovine Tissues in Food and Feed

Journal of Food Protection ◽

10.4315/0362-028x-71.3.564 ◽

2008 ◽

Vol 71 (3) ◽

pp. 564-572 ◽

Cited By ~ 11

Author(s):

IRENE MARTÍN ◽

TERESA GARCÍA ◽

VIOLETA FAJARDO ◽

MARÍA ROJAS ◽

PABLO E. HERNÁNDEZ ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

18S Rrna ◽

Detection System ◽

Total Content ◽

18S Rrna Gene ◽

Universal Primers ◽

Quantitative Detection ◽

12S Rrna ◽

Rrna Gene

A real-time PCR approach with the SYBR Green detection system has been developed for the quantitative detection of bovine tissues in food and feedstuffs. The method combines the use of bovine-specific primers, which amplify an 84-bp fragment of the mitochondrial 12S rRNA gene, and universal primers, which amplify a 140-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. The 18S rRNA primers are used as endogenous controls for the total content of PCR-amplifiable DNA in the sample. The specificity of the primers was tested against 18 animal species, including mammals, birds, and fish, as well as 6 plant species. Analysis of experimental bovine tissues–oats mixtures demonstrated the suitability of the assay for the detection of bovine DNA in mixtures containing as low as 0.1% of bovine tissues. The performance of the method is not affected by severe heat treatment (up to 133°C for 20 min at 300 kPa). The reported PCR assay could be very useful for detecting bovine-derived ingredients in raw and heat-treated food and feedstuffs.

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Sensitivity of multiplex real-time PCR reactions, using the LightCycler and the ABI PRISM 7700 Sequence Detection System, is dependent on the concentration of the DNA polymerase

Molecular and Cellular Probes ◽

10.1006/mcpr.2002.0433 ◽

2002 ◽

Vol 16 (5) ◽

pp. 351-357 ◽

Cited By ~ 15

Author(s):

M.M. Exner ◽

M.A. Lewinski

Keyword(s):

Real Time ◽

Dna Polymerase ◽

Real Time Pcr ◽

Detection System ◽

Sequence Detection

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Detecting human cytomegalovirus drug resistant mutations and monitoring the emergence of resistant strains using real-time PCR

Journal of Clinical Virology ◽

10.1016/j.jcv.2014.07.008 ◽

2014 ◽

Vol 61 (2) ◽

pp. 270-274 ◽

Cited By ~ 5

Author(s):

Pavlina Volfova ◽

Martina Lengerova ◽

Jana Lochmanova ◽

Dana Dvorakova ◽

Dita Ricna ◽

...

Keyword(s):

Real Time ◽

Human Cytomegalovirus ◽

Real Time Pcr ◽

Drug Resistant ◽

Resistant Strains

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Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

Microorganisms ◽

10.3390/microorganisms9051031 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1031

Author(s):

Roberto Zoccola ◽

Alessia Di Blasio ◽

Tiziana Bossotto ◽

Angela Pontei ◽

Maria Angelillo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Detection System ◽

Bacterial Load ◽

Microbial Control ◽

Hospital Setting ◽

Sample Volume ◽

Analytical Sensitivity ◽

Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

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Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3911-3916.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3911-3916 ◽

Cited By ~ 71

Author(s):

Mark G. Wise ◽

Gregory R. Siragusa

Keyword(s):

Gastrointestinal Tract ◽

Real Time ◽

Clostridium Perfringens ◽

Real Time Pcr ◽

Limit Of Detection ◽

Quantitative Detection ◽

Necrotic Enteritis ◽

Analytical Sensitivity ◽

Quantitative Real Time Pcr ◽

Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

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