scholarly journals Comparison of real‐time PCR and cultural method for detection of bacterial load in pasteurized milk

2019 ◽  
Vol 39 (3) ◽  
pp. e12624 ◽  
Author(s):  
Ayda Farhoudi ◽  
Peyman Ghajarbeygi ◽  
Razi Allah Jafari Jozani ◽  
Razzagh Mahmoudi ◽  
Karim Mardani
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Load ◽  
Pasteurized Milk ◽  
Cultural Method

scholarly journals 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

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.

O-091 Semen microbiota in patients with astenozoospermia and healthy controls: cluster analysis of real-time PCR data

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Panacheva ◽  
D Pochernikov ◽  
E Voroshilina
Keyword(s):  
Cluster Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Detection Rate ◽  
Bacterial Load ◽  
Rrna Gene ◽  
Rt Pcr ◽  
Silhouette Index ◽  
Enterococcus Spp ◽  
Lactobacillus Spp

Abstract Study question What are the differences in the semen microbiota composition of patients with asthenozoospermia and normospermia according to cluster analysis of PCR data? Summary answer The detection rate of 4 stable semen microbiota clusters and the dominant bacteria groups varied in patients with asthenozoospermia and normospermia. What is known already Most of the research dedicated to analyzing normal and pathological semen microbiota is based on 16S rRNA gene specific Next generation sequencing (NGS). It has shown that microbiota is represented by polymicrobial communities (clusters) that consist of microorganisms from different genera and bacteria phyla. Despite it being highly informative, NGS has several weaknesses: complex sample preparation, difficult sample intake control, long analysis process, complicated results interpretation, high cost of equipment and reagents. These factors make it virtually impossible to use this approach in routine medical practice. Quantitative real-time PCR (RT-PCR) is far more suitable for this. Study design, size, duration Patients included in the study (n = 301) came to the “Garmonia” Medical Center (Yekaterinburg, Russia) either seeking preconception care or for infertility treatment. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 171) — asthenozoospermia, Group 2 (n = 130) — normospermia. Participants/materials, setting, methods Semen microbiota was analyzed using RT-PCR kit Androflor (DNA-Technology, Russia). Cluster analysis was performed for 201 samples with the total bacterial load (TBL) of at least 103 GE/ml (asthenozoospermia = 96, normospermia = 105). Cluster analysis was conducted using the k-means ++ algorithm, scikit-learn. The Silhouette index and the Davies–Bouldin index (DBI) were used to confirm the stability of clusters. Main results and the role of chance Both in the samples with normospermia and asthenozoospermia, four stable microbiota clusters were distinguished. Cluster I was characterized by the prevalence of obligate anaerobes, Lactobacillus spp. were prevalent in Cluster II, Gram-positive facultative anaerobes were prevalent in Cluster III, Enterobacteriaceae/Enterococcus spp. were prevalent in Cluster IV. Cluster I was detected the most often in both groups. However, in normospermia it was represented by various obligate anaerobes without pronounced quantitative predominance of any bacteria group. In samples with asthenozoospermia one of the bacteria groups were prevalent in Cluster I: Bacteroides spp./Porphyromonas spp./Prevotella spp., Peptostreptococcus spp./Parvimonas spp. or Eubacterium spp. In samples with asthenozoospermia Cluster II was characterized by the prevalence of Lactobacillus spp., while in samples with normospermia other bacteria groups were present along with lactobacilli, mainly obligate anaerobes. In samples with normospermia Corynebacterium spp. and Streptococcus spp., typical of normal microbiota of male UGT, were prevalent in Cluster III. In samples with asthenozoospermia Cluster III were characterized by the prevalence of Staphylococcus spp. In samples with asthenozoospermia Lactobacillus spp was present in Cluster IV along with Enterobacteriaceae/Enterococcus spp., which was not typical of the samples with normospermia. Limitations, reasons for caution Cluster analysis was not conducted for the samples with TBL lower than 103 GE/ml, since their results were incompatible with the data received for the negative control samples. Wider implications of the findings Further research could determine the detection rate of the described bacterial clusters in semen with other pathologies. Establishing the relationship between the characteristics of semen microbiota and infertility in men might allow the development of new algorithms for treating patients with reproductive disorders, depending on the composition of semen microbiota. Trial registration number not applicable

scholarly journals Detection and quantification of Campylobacter in foods: New analytic approaches to detect and quantify Campylobacter spp. in food samples

2020 ◽  
Vol 9 (2) ◽  
Author(s):  
Maria Francesca Peruzy ◽  
Yolande Thérèse Rose Proroga ◽  
Federico Capuano ◽  
Federica Corrado ◽  
Serena Santonicola ◽  
...  
Keyword(s):  
Real Time ◽  
Campylobacter Jejuni ◽  
Internal Control ◽  
Real Time Pcr ◽  
Quantitative Methods ◽  
Bacterial Load ◽  
Digital Pcr ◽  
Food Samples ◽  
Meat Samples ◽  
Campylobacter Spp

The aim of the present study was to develop rapid qualitative and quantitative methods based on the use of Real-Time PCR and Droplet Digital PCR (ddPCR), in order to have reliable techniques to detect and quantify Campylobacter spp. in food samples. The gene 16S-rRNA was used as specific target for Campylobacter spp. Real- Time PCR evaluation assay and a not competitive internal control was ushered in it. To investigate the selectivity of the method, 26 Campylobacter strains and 40 non-Campylobacter strains were tested and in order to verify the application of Real-Time PCR method, 5 pork meat samples were experimentally inoculated with a Campylobacter jejuni strain. Subsequently, dilutions with a bacterial load of Campylobacter jejuni within 10-106 CFU/mL were chosen for the optimization of the ddPCR assay. Lastly, a total of 54 naturally contaminated foods samples were analyzed through molecular (Real-Time PCR and ddPCR) and traditional methods. The Real-Time PCR protocol demonstrated to amplify only the Campylobacter spp. strains and when Campylobacter jejuni was experimentally inoculated in meat samples the pathogen was always detected. The ddPCRs assay allowed to quantify a level of contamination of 10 CFU/mL, but it was unable to quantify levels of 105 – 106 CFU/mL. Lastly, Campylobacter spp. was never detected in the 54 samples tested. In conclusion, the novel analytic approach proposed, based on an initial screening of the samples with Real-Time PCR and then on quantification of Campylobacter spp. with a ddPCR on those positive, represents a quick monitoring tool and, if used correctly, it would allow the implementation of food safety.

scholarly journals Evaluation of a Multiplex Real-Time PCR Assay for Detecting Major Bacterial Enteric Pathogens in Fecal Specimens: Intestinal Inflammation and Bacterial Load Are Correlated in Campylobacter Infections

2016 ◽  
Vol 54 (9) ◽  
pp. 2262-2266 ◽  
Author(s):  
Nadia Wohlwend ◽  
Sacha Tiermann ◽  
Lorenz Risch ◽  
Martin Risch ◽  
Thomas Bodmer
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Load ◽  
Positive Culture ◽  
Fecal Calprotectin ◽  
Enteric Pathogens ◽  
Significant Linear Trend ◽  
Content Type ◽  
Culture Positive ◽  
Culture Negative

A total of 1,056 native or Cary-Blair-preserved stool specimens were simultaneously tested by conventional stool culturing and by enteric bacterial panel (EBP) multiplex real-time PCR forCampylobacter jejuni,Campylobacter coli,Salmonellaspp., and shigellosis disease-causing agents (Shigellaspp. and enteroinvasiveEscherichia coli[EIEC]). Overall, 143 (13.5%) specimens tested positive by PCR for the targets named above; 3 coinfections and 109 (10.4%)Campylobacterspp., 17 (1.6%)Salmonellaspp., and 20 (1.9%)Shigellaspp./EIEC infections were detected. The respective positive stool culture rates were 75 (7.1%), 14 (1.3%), and 7 (0.7%). The median threshold cycle (CT) values of culture-positive specimens were significantly lower than those of culture-negative ones (CTvalues, 24.3 versus 28.7;P< 0.001), indicating that the relative bacterial load per fecal specimen was significantly associated with the culture results. InCampylobacterinfections, the respective median fecal calprotectin concentrations in PCR-negative/culture-negative (n =40), PCR-positive/culture-negative (n =14), and PCR-positive/culture-positive (n =15) specimens were 134 mg/kg (interquartile range [IQR], 30 to 1,374 mg/kg), 1,913 mg/kg (IQR, 165 to 3,813 mg/kg), and 5,327 mg/kg (IQR, 1,836 to 18,213 mg/kg). Significant differences were observed among the three groups (P< 0.001), and a significant linear trend was identified (P< 0.001). Furthermore, the fecal calprotectin concentrations andCTvalues were found to be correlated (r= −0.658). Our results demonstrate that molecular screening ofCampylobacterspp.,Salmonellaspp., andShigellaspp./EIEC using the BD Max EBP assay will result in timely diagnosis and improved sensitivity. The determination of inflammatory markers, such as calprotectin, in fecal specimens may aid in the interpretation of PCR results, particularly for enteric pathogens associated with mucosal damage and colonic inflammation.

scholarly journals Searching for Bacterial Pathogens in Pediatric Patients with Chronic Recurrent Multifocal Osteomyelitis Using 16S rRNA Quantitative Real-Time PCR

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S98-S98
Author(s):  
Justin Searns
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Pathogens ◽  
Bacterial Load ◽  
Chronic Recurrent Multifocal Osteomyelitis ◽  
Molecular Diagnostic ◽  
Quantitative Real Time Pcr ◽  
Bone Biopsies ◽  
Multifocal Osteomyelitis

Abstract Background Chronic recurrent multifocal osteomyelitis (CRMO) is a rare auto-inflammatory disease in children that causes relapsing episodes of pain. Patients are treated with anti-inflammatory medications or immune-modulating agents. Increasing evidence suggests that CRMO is mediated by dysregulation of the interleukin-1 pathway, not a bacterial source. However, CRMO is often a diagnosis of exclusion, and patients occasionally receive antimicrobials for possible culture negative infectious osteomyelitis. Few prior studies have utilized molecular diagnostic techniques to identify bacterial pathogens in CRMO bone biopsies. Methods Musculoskeletal specimens sent for culture during routine clinical care were banked from patients admitted to Children’s Hospital Colorado from 6/2012 to 10/2016. On retrospective chart review, 28 specimens were collected from 16 patients ultimately diagnosed with CRMO. Specimens were processed and extracted prior to molecular testing. All samples underwent quantitative real-time PCR (qPCR) testing using bacterial load assays targeting the bacterial 16S rRNA gene. Results Mean age at time of sample collection was 9.2 years. CRMO diagnosis was made by clinical, pathologic, and radiographic findings. All patients had pathology findings consistent with CRMO including lymphoplasmacytic infiltrate, focal necrosis, and/or marrow fibrosis. All patients had MRI findings consistent with CRMO. No patient had bacteria identified on Gram stain; 2/28 samples (7%) had bacterial growth on culture (both were coagulase-negative staphylococcus, felt to be contaminant). None of the 28 specimens met the threshold of bacterial load on qPCR testing to necessitate bacterial sequencing. None of the 16 patients were treated with antimicrobials and there were no readmissions for clinical worsening. Conclusion CRMO patients did not have bacteria identified on universal bacterial 16S rRNA testing. This finding further supports that CRMO patients do not require antimicrobial therapy. Future steps to exclude infectious pathogens in CRMO could include next-generation DNA sequencing. Disclosures All authors: No reported disclosures.

scholarly journals Validation of a Commercial Loop-Mediated Isothermal Amplification (LAMP)-Based Kit for the Detection of Salmonella spp. According to ISO 16140:2016

2021 ◽  
Vol 11 (15) ◽  
pp. 6669
Author(s):  
Calogero Di Bella ◽  
Antonella Costa ◽  
Sonia Sciortino ◽  
Giuseppa Oliveri ◽  
Gaetano Cammilleri ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Method ◽  
Poultry Meat ◽  
Lamp Method ◽  
Salmonella Spp ◽  
Cultural Method ◽  
Detection And Identification ◽  
Biochemical Identification ◽  
Raw Poultry

The traditional cultural method (PCR and Real-Time PCR) for Salmonella spp. detection and identification is laborious and time-consuming. A qualitative LAMP method detecting Salmonella spp. was validated in compliance with ISO 16140:2016. The results show a relative accuracy, sensitivity, and specificity of 100% in comparison with the reference method ISO 6579-1:2017; the LOD50 was set as 0.4 CFU/g. Additionally, a field study was carried out comparing the LAMP kit, a commercially available Real-Time PCR kit (FoodProof Salmonella, Biotecon Diagnostics), and the reference cultural method. The Salmonella spp. LAMP kit was suitable for reliable detection of Salmonella spp., simplifying and reducing the extent and the steps of the analytical process. A total of 105 samples of raw poultry meat were screened for the presence of Salmonella spp. according to three methods: the LAMP kit Salmonella spp. (Enbiotech), the Real-Time PCR kit FoodProof Salmonella (Biotecon), and the reference cultural method. Using these three methods, only one sample out of the 105 (0.95%) tested was positive for Salmonella spp. This sample was further investigated using the reference method described in ISO 6579-3:2014, in order to characterise the Salmonella strain. Following this further biochemical identification and serological typing, the isolate was characterised as Salmonella Infantis.

scholarly journals Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set

Microbiology ◽  
2002 ◽  
Vol 148 (1) ◽  
pp. 257-266 ◽  
Author(s):  
Mangala A Nadkarni ◽  
F. Elizabeth Martin ◽  
Nicholas A Jacques ◽  
Neil Hunter
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Load

scholarly journals Characterization and Modeling of Salmonella Growth in Pasteurized and Non-pasteurized Milk Using Real-Time PCR

2014 ◽  
Vol 31 (1) ◽  
pp. 28-35 ◽  
Author(s):  
Susumu KAWASAKI ◽  
Shigemasa SHIMIZU ◽  
Shigenobu KOSEKI ◽  
Yasuhiro INATSU
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pasteurized Milk
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