Evaluation of New Preanalysis Sample Treatment Tools and DNA Isolation Protocols To Improve Bacterial Pathogen Detection in Whole Blood

Hansen, Wendy L. J.; Bruggeman, Cathrien A.; Wolffs, Petra

doi:10.1128/jcm.00821-09

Cited by 48 publications

(36 citation statements)

References 11 publications

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“…Minimal handling was also favorable with regard to implementation of the rapid method in a high-throughput routine laboratory. The use of enzymes and bile products for selective removal of interacting sample material has previously been reported (28,31). Although those studies were based on samples of whole blood, the challenges are similar to those of the present study, i.e., high levels of PCR inhibitors and very low concentration of the targeted pathogen.…”

Section: Discussionmentioning

confidence: 50%

“…Studies have shown that PCR inhibitors can be reduced by physical, biochemical, immunological, and physiological sample preparation methods. These include flotation (18), filtration (9,20), adsorption and addition of surfactants (19), enzymatic digestion (9), DNA extraction (28), and enrichment in a PCR-compatible medium (15), all of which were considered when designing the described rapid method.…”

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confidence: 99%

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Detection of Salmonella enterica in Meat in Less than 5 Hours by a Low-Cost and Noncomplex Sample Preparation Method

Fachmann

Löfström

Hoorfar

et al. 2017

Appl Environ Microbiol

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Salmonella is recognized as one of the most important foodborne bacteria and has wide health and socioeconomic impacts worldwide. Fresh pork meat is one of the main sources of Salmonella, and efficient and fast methods for detection are therefore necessary. Current methods for Salmonella detection in fresh meat usually include Ͼ16 h of culture enrichment, in a few cases Ͻ12 h, thus requiring at least two working shifts. Here, we report a rapid (Ͻ5 h) and high-throughput method for screening of Salmonella in samples from fresh pork meat, consisting of a 3-h enrichment in standard buffered peptone water and a real-time PCR-compatible sample preparation method based on filtration, centrifugation, and enzymatic digestion, followed by fast-cycling real-time PCR detection. The method was validated in an unpaired comparative study against the Nordic Committee on Food Analysis (NMKL) reference culture method 187. Pork meat samples (n ϭ 140) were either artificially contaminated with Salmonella at 0, 1 to 10, or 10 to 100 CFU/25 g of meat or naturally contaminated. Cohen's kappa for the degree of agreement between the rapid method and the reference was 0.64, and the relative accuracy, sensitivity, and specificity for the rapid method were 81.4, 95.1, and 97.9%, respectively. The 50% limit of detections (LOD 50 s) were 8.8 CFU/25 g for the rapid method and 7.7 CFU/25 g for the reference method. Implementation of this method will enable faster release of Salmonella low-risk meat, providing savings for meat producers, and it will help contribute to improved food safety.IMPORTANCE While the cost of analysis and hands-on time of the presented rapid method were comparable to those of reference culture methods, the fast product release by this method can provide the meat industry with a competitive advantage. Not only will the abattoirs save costs for work hours and cold storage, but consumers and retailers will also benefit from fresher meat with a longer shelf life. Furthermore, the presented sample preparation might be adjusted for application in the detection of other pathogenic bacteria in different sample types.KEYWORDS Salmonella, food safety, foodborne pathogens, fresh meat, rapid tests, real-time PCR, sample preparation, validation S almonella is a global foodborne pathogen responsible for approximately 30% of foodborne outbreaks in the United States and 23% in the European Union (1, 2). Pork meat remains an important source of human salmonellosis, which is why the absence of Salmonella is confirmed at critical control points at abattoirs (2). However, the reference culture-based methods require several days to obtain a test result (3, 4), thereby postponing the market release of fresh meat.

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Section: Discussionmentioning

confidence: 50%

mentioning

confidence: 99%

Detection of Salmonella enterica in Meat in Less than 5 Hours by a Low-Cost and Noncomplex Sample Preparation Method

Fachmann

Löfström

Hoorfar

et al. 2017

Appl Environ Microbiol

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“…This can be very challenging if genomic DNA is copurified with targeted microbial DNA (11)(12)(13). A number of solutions to this problem have been proposed (14)(15)(16)(17)(18)(19) but, to date, none have achieved the desired sensitivity (20). It should be noted that many of these methods do appear to capture a significantly higher number of total positives than culture, and the "extra" detections are often correlated with clinical indications of bloodstream infection; however, an inability to capture all positives detected by culture (false negatives) has been an issue.…”

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confidence: 99%

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

et al. 2014

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The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml wholeblood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

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“…Furthermore, the reduced burden of human DNA is likely to improve the sensitivity and specificity of pathogen detection compared to other tests that are based on the isolation of total DNA from CSF samples. Using blood samples spiked with methicillinresistant Staphylococcus aureus (MRSA), bacterial DNA was detected by PCR with higher sensitivity in DNA extracts obtained with the preanalytical UMD-Liquid system than in total DNA extracts (16). Improved sensitivity over conventional diagnostics was also shown in clinical studies on patients with endocarditis, sepsis, and joint infections (12,17,18).…”

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confidence: 99%

Improved Detection of Bacterial Central Nervous System Infections by Use of a Broad-Range PCR Assay

et al. 2014

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Evaluation of New Preanalysis Sample Treatment Tools and DNA Isolation Protocols To Improve Bacterial Pathogen Detection in Whole Blood

Cited by 48 publications

References 11 publications

Detection of Salmonella enterica in Meat in Less than 5 Hours by a Low-Cost and Noncomplex Sample Preparation Method

Detection of Salmonella enterica in Meat in Less than 5 Hours by a Low-Cost and Noncomplex Sample Preparation Method

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

Improved Detection of Bacterial Central Nervous System Infections by Use of a Broad-Range PCR Assay

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