scholarly journals Quantification of Enterococci and Human Adenoviruses in Environmental Samples by Real-Time PCR

2005 ◽  
Vol 71 (5) ◽  
pp. 2250-2255 ◽  
Author(s):  
Jian-Wen He ◽  
Sunny Jiang
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Bacterial Culture ◽  
Culture Method ◽  
23S Rrna ◽  
Dilution Series ◽  
Human Adenoviruses ◽  
Standard Curve ◽  
Serial Dilutions

ABSTRACT Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.

An improved real-time qPCR technique for quantification of intestinal bacteria in human fecal samples

2018 ◽  
Vol 7 (5) ◽  
pp. 201-209
Author(s):  
Sama Rezasoltani ◽  
Hossein Dabiri ◽  
Hamid Asadzadeh Aghdaei ◽  
Abbas Akhavan Sepahi ◽  
Mohammad Hossein Modarressi ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
Intestinal Bacteria ◽  
Fecal Samples ◽  
Standard Curve ◽  
Dissociation Curves ◽  
Real Time Qpcr ◽  
Serial Dilutions

The easy, cost-effective and rapid techniques for investigating the role of gut microbiota have become imperative owing to their importance in various diseases such as colon cancer and inflammatory bowel disease. Here we developed an absolute real-time qPCR technique for quantification of intestinal bacteria in human fecal samples for further investigation of gut microbiota composition in patients. At first, regarding bacterial species present in human fecal samples, species were cultured in anaerobic culture media and incubated at 37Cͦ in an anaerobic chamber. The number of CFU was counted. Eight fold serial dilutions of the bacterial suspension were prepared. To generate a standard curve for bacterial species in real-time PCR, DNA was extracted from different serial dilutions. Subsequently, DNA from human fecal samples was extracted. Now bacterial strains were applied as standard curve in absolute q PCR procedure for quantification of bacterial population in test samples. Based on absolute real-time analysis, intestinal bacteria were precisely quantified. The specificity of the primer pairs was checked by conventional PCR and real-time PCR regarding dissociation curve analysis. The amplification was linear in the range of 101-108. Dissociation curves had correlation coefficient values between 0.98 and 101/888. The efficiencies were between 95/073 and 99/804%. The absolute real-time PCR technique described here may be used for evaluating intestinal bacteria regarding diseases prevention.

A real-time PCR regimen for testing environmental samples for Salmonella enterica subsp. enterica serovars of concern to the poultry industry, with special focus on Salmonella Enteritidis

2019 ◽  
Vol 65 (2) ◽  
pp. 162-173 ◽  
Author(s):  
S. Nadin-Davis ◽  
L. Pope ◽  
D. Ogunremi ◽  
B. Brooks ◽  
J. Devenish
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Culture Collection ◽  
Special Focus ◽  
Culture Methods ◽  
Poultry Facilities ◽  
Culture Process

A real-time PCR (qPCR) regimen, using up to six genetic targets, was developed to rapidly detect Salmonella and in particular identify Salmonella Enteritidis. The test regimen was first evaluated using a reference culture collection of Salmonella to confirm the appropriateness of the selected targets, which included up to three genetic markers for discrimination of Salmonella Enteritidis from other Salmonella serovars commonly found in poultry facilities. The qPCR procedure was then compared with culture methods used to detect Salmonella using a collection of enrichment broths previously generated from 239 environmental samples collected from a large number of hatchery facilities across Canada over several years. The qPCR regimen facilitated specific detection of Salmonella Enteritidis, and on a sample basis, it showed excellent agreement with the culture methods. Moreover, in many cases, qPCR detected Salmonella earlier in the culture process than did the culture method. Application of this method will significantly shorten test times and allow more timely identification of infected poultry premises, thereby improving present programmes aimed at controlling Salmonella Enteritidis at the environmental source.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

scholarly journals Evaluation of StrepBSelectChromogenic Medium and the Fast-Track Diagnostics Group B Streptococcus (GBS) Real-Time PCR Assay Compared to Routine Culture for Detection of GBS during Antepartum Screening

2017 ◽  
Vol 55 (7) ◽  
pp. 2137-2142 ◽  
Author(s):  
Deirdre L. Church ◽  
Heather Baxter ◽  
Tracie Lloyd ◽  
Oscar Larios ◽  
Daniel B. Gregson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fast Track ◽  
Group B Streptococcus ◽  
Culture Method ◽  
Predictive Values ◽  
Content Type ◽  
Standard Culture ◽  
Group B ◽  
The Cost

ABSTRACTLife-threatening infection in neonates due to group BStreptococcus(GBS) is preventable by screening of near-term pregnant women and treatment at delivery. A total of 295 vaginal-rectal swabs were collected from women attending antepartum clinics in Calgary, Alberta, Canada. GBS colonization was detected by the standard culture method (Strep B Carrot Broth subcultured to blood agar with a neomycin disk) and compared to recovery with Strep Group B Broth (Dalynn Biologicals) subcultured to StrepBSelectchromogenic medium (CM; Bio-Rad Laboratories) and the Fast-Track Diagnostics GBS real-time PCR (quantitative PCR [qPCR]) assay (Phoenix Airmid Biomedical Corp.) performed with broth-enriched samples and the Abbottm2000sp/m2000rt system. A total of 62/295 (21%) women were colonized with GBS; 58 (19.7%) cases were detected by standard culture, while CM and qPCR each found 61 (20.7%) cases. The qPCR and CM were similar in performance, with sensitivities, specificities, and positive and negative predictive values of 98.4 and 98.4%, 99.6 and 99.6%, 98.4 and 98.4%, and 99.6 and 99.6%, respectively, compared to routine culture. Both qPCR and CM would allow more rapid reporting of routine GBS screening results than standard culture. Although the cost per test was similar for standard culture and CM, the routine use of qPCR would cost approximately four times as much as culture-based detection. Laboratories worldwide should consider implementing one of the newer methods for primary GBS testing, depending on the cost limitations of different health care jurisdictions.

Detection and Quantification of Human Adenoviruses in Surface Waters by Nested PCR, TaqMan Real-Time PCR and Cell Culture Assays

2007 ◽  
Vol 191 (1-4) ◽  
pp. 83-93 ◽  
Author(s):  
M. Muscillo ◽  
M. Pourshaban ◽  
M. Iaconelli ◽  
S. Fontana ◽  
A. Di Grazia ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Surface Waters ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Human Adenoviruses ◽  
Detection And Quantification

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

scholarly journals Isolation and molecular characterization of Mycobacterium tuberculosis complex isolated from raw milk in some dairy farms in Egypt

2016 ◽  
Vol 5 (2) ◽  
pp. 105
Author(s):  
Heba Hussien ◽  
Eman Mahrous
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Raw Milk ◽  
Accurate Method ◽  
Tuberculosis Complex ◽  
Milk Samples ◽  
Source Of Infection

<p>This study was conducted to detect <em>Mycobacterium tuberculosis</em> complex in milk in three Egyptian Governorates; El-Sharkia, El-Menoufia and El-Behera Governorates. 300 milk samples were collected from tuberculin positive cases, 18 (6.0%) were shedding <em>Mycobacterium tuberculosis</em> complex in their milk which detected by real time PCR. On another hand, 170 milk samples were collected from tuberculin negative cases, 5 (2.9%) were shedding <em>Mycobacterium tuberculosis</em> complex in their milk which detected by real time PCR. All milk samples were examined by three techniques including Microscopic examination, culture and real time PCR. Real time PCR is more rapid and accurate method than microscopic and culture method. The isolated colonies from culture were examined by Multiplex PCR to demonstrate the source of infection either human or animal source.</p>

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