Use of functional genes to quantify denitrifiers in the environment

2006 ◽  
Vol 34 (1) ◽  
pp. 101-103 ◽  
Author(s):  
L. Philippot
Keyword(s):  
Real Time Pcr ◽  
Southern Hybridization ◽  
Most Probable Number ◽  
Functional Genes ◽  
Hybridization Technique ◽  
Dna Arrays ◽  
Competitive Pcr ◽  
Probable Number ◽  
Composition And Structure ◽  
Micro Organisms

During the last decade, application of molecular methods using cultivation-independent approaches has provided new insights into the composition and structure of denitrifying communities in various environments. However, little is known about their abundance, and quantification is still performed using cultivation-based approaches, which are not only biased by the inability to cultivate of many micro-organisms but also fastidious and time-consuming. Two types of cultivation-independent approaches have recently been developed to quantify denitrifiers. The first type, which is based on the hybridization technique, comprises the use of Southern hybridization and DNA arrays. The second type, based on PCR, comprises the use of MPN (most probable number)-PCR, competitive PCR or real-time PCR. In this review, these different approaches will be presented with examples of their application in environmental studies.

Development of Rapid Real-Time PCR and Most-Probable-Number Real-Time PCR Assays To Quantify Enterotoxigenic Strains of the Species in the Bacillus cereus Group

2007 ◽  
Vol 70 (12) ◽  
pp. 2774-2781 ◽  
Author(s):  
I-CHEN YANG ◽  
DANIEL YANG-CHIH SHIH ◽  
JAN-YI WANG ◽  
TZU-MING PAN
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Samples ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Bacillus Cereus Group ◽  
Probable Number ◽  
Cooked Rice ◽  
Group Detection

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (102 to 107 CFU/g for cooked rice and chicken, 103 to 107 CFU/ml for milk, and 104 to 107 CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 100 CFU/ml. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification.

scholarly journals Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters

2007 ◽  
Vol 73 (18) ◽  
pp. 5840-5847 ◽  
Author(s):  
Jessica L. Nordstrom ◽  
Michael C. L. Vickery ◽  
George M. Blackstone ◽  
Shelley L. Murray ◽  
Angelo DePaola
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
The United States ◽  
Internal Amplification Control ◽  
Most Probable Number ◽  
Specific Marker ◽  
Pcr Assay ◽  
Probable Number ◽  
The Real

ABSTRACT Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >104 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh + and trh + strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.

scholarly journals Bacteriological Quality of Drinking Water in Mardan, Khyber Pakhtunkhwa, Pakistan

2015 ◽  
Vol 21 ◽  
pp. 1-6
Author(s):  
Javid Ali ◽  
Said Hassan ◽  
Dr Ziaurahman ◽  
Inayat Ur Rahman ◽  
Sadhair Abbas ◽  
...  
Keyword(s):  
Water Quality ◽  
Drinking Water ◽  
Fecal Coliforms ◽  
Plate Count ◽  
Most Probable Number ◽  
Probable Number ◽  
Khyber Pakhtunkhwa ◽  
E Coli ◽  
Micro Organisms

The present study was aimed to isolate and identify micro-organisms load of drinking water of Mardan city, Khyber Pakhtunkhwa Pakistan. A total of 27 samples of drinking water were collected from different locations of the study area. Total Plate Count was determined by pour plate method, while total coliforms, total fecal coliforms and E. coli were determined by multiple tube fermentation method. Of the total collected samples, 17 (62.96%) samples were contaminated with either one or more than one type of microorganisms. The results of most probable number test showed that 13 (48.15%) samples were unsatisfactory. It was concluded that the water should be treated before consumption for drinking purpose. Regular assessment of the water quality is recommended as regular monitoring of the water quality for improvement not only prevents disease and hazards but also checks the water resources from becoming further polluted. ECOPRINT 21: 1-6, 2014DOI: http://dx.doi.org/10.3126/eco.v21i0.11897

Detection and Quantification of Campylobacter in Poultry Slaughterhouses Using Conventional Microbiological Technique, Most Probable Number, and Real-Time PCR

2021 ◽  
Author(s):  
Gustavo Perdoncini ◽  
Yuli Melisa Sierra Arguello ◽  
Leonardo Moreira Lima ◽  
Thales Quedi Furian ◽  
Karen Apellanis Borges ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Most Probable Number ◽  
Probable Number ◽  
Detection And Quantification

Development of a Quantitative Real-Time PCR Method for Estimation of the Total Number of Vibrio parahaemolyticus in Contaminated Shellfish and Seawater

2005 ◽  
Vol 68 (5) ◽  
pp. 1083-1088 ◽  
Author(s):  
HAJIME TAKAHASHI ◽  
YOSHITO IWADE ◽  
HIROTAKA KONUMA ◽  
YUKIKO HARA-KUDO
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pure Culture ◽  
Vibrio Parahaemolyticus ◽  
Plate Count ◽  
Most Probable Number ◽  
Cycle Number ◽  
Probable Number ◽  
The Real ◽  
Pcr Method

A real-time PCR method targeting the toxR gene of Vibrio parahaemolyticus was developed to quantify the number of V. parahaemolyticus cells, including those of both the hemolysin-producing and nonproducing strains. The specificity of the primer and probe set was confirmed using 25 strains of V. parahaemolyticus and 30 strains of other microbial species. We determined the threshold cycle number using the real-time PCR and the number of V. parahaemolyticus cells by plate count using serially diluted pure culture and developed a standard curve for quantification. Standard curves for V. parahaemolyticus in seawater and seafood were established using artificially inoculated samples. The threshold cycle number and the number of V. parahaemolyticus cells were correlated with 101 to 107 CFU/ml in pure culture, seawater, and shellfish homogenate. The real-time PCR method developed in this study was compared with the most-probable-number method in seafood samples that were naturally contaminated. The differences in the number of V. parahaemolyticus cells as determined by the culture method and the PCR method were less than 10-fold.

scholarly journals Βιοαντιδραστήρες μεμβρανών για την επεξεργασία βιομηχανικών λυμάτων

2014 ◽  
Author(s):  
Παναγιώτα Μπαμπατσούλη
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Most Probable Number ◽  
Ammonia Oxidizing Bacteria ◽  
Probable Number

Στο α μέρος της παρούσας διατριβής μελετάται μια πιλοτική μονάδα βιολογικού καθαρισμού με βιοαντιδραστήρα μεμβρανών, η οποία επεξεργάζεται τα βιομηχανικά απόβλητα που εισέρχονται στις εγκαταστάσεις του βιολογικού της ΒΙ.ΠΕ Ηρακλείου Κρήτης, χωρίς καμμία προεπεξεργασία, πέραν από αυτή της εσχάρωσης. Το συγκεκριμένο απόβλητο χαρακτηρίζεται κυρίως από το πολύ υψηλό οργανικό φορτίo που περιέχει και τις υψηλές διακυμάνσεις στη σύστασή του. Οι αναλύσεις που πραγματοποιήθηκαν κατά τη διάρκεια της διατριβής στο συγκεκριμένο σύστημα αφορούσαν α) την εξέταση της επίδρασης του χρόνου παραμονής της λάσπης στην ποιότητα της εκροής β) τον προσδιορισμό των διαλυτών και δεσμευμένων εξωκυτταρικών πολυμερών που παράγονται στο σύστημα με στόχο την διερεύνηση της επίδρασης του χρόνου παραμονής της λάσπης στην έμφραξη των μεμβρανών και γ) την εξέταση της επίδρασης του υδραυλικού χρόνου παραμονής τόσο στην ποιότητα εξόδου της εκροής, όσο και στη συσσώρευση των εξωκυτταρικών πολυμερών ενώσεων και των διαλυτών μικροβιακών προϊόντων, σε σταθερό χρόνο παραμονής της λάσπης.Επιπλέον στα πλαίσια αυτής της διατριβής και στα ευρύτερα πλαίσια του όρου ‘βιοαντιδραστήρες μεμβρανών’ στο β μέρος εξετάστηκε ένας αντιδραστήρας προσκολλημένης βιομάζας χρησιμοποιώντας ως μέσο ανάπτυξης των μικροοργανισμών ένα πατενταρισμένο ύφασμα με στόχο τη μελέτη της επεξεργασίας ενός συνθετικού αποβλήτου που προσομοιώνει το ‘βιομηχανικό’ απόβλητο που προέρχεται από την έξοδο θαλασσινών υδατοκαλλιεργειών. Με την πάροδο του χρόνου αναπτύχθηκαν βακτήρια πάνω στο ύφασμα του αντιδραστήρα και κατόπιν πραγματοποιήθηκε ο εμβολιασμός του συστήματος με συγκεκριμένο γένος μικροαλγών για την αποτελεσματικότερη λειτουργία του. Οι αναλύσεις οι οποίες πραγματοποιήθηκαν στο συγκεκριμένο σύστημα είχαν ως στόχο: α) τον προσδιορισμό του βέλτιστου χρόνου λειτουργίας της αντλίας του αντιδραστήρα β) την περιγραφή της μικροβιακής κοινότητας που αποτελούσε το βιοφίλμ που αναπτύχθηκε στο ύφασμα του αντιδραστήρα και που προσδιορίστηκε συγκεκριμένα με τη μέθοδο του Pyrosequencing. γ) τον προσδιορισμό του αριθμού των συνολικών κυττάρων των μικροαλγών και των βακτηρίων ανά τετραγωνικό εκατοστό βιοφίλμ που εκτιμήθηκε με βάση τον αλγόριθμο του πιο πιθανού αριθμού (Most Probable Number - MPN) δ) την αποδοτικότητα του συστήματος (όσον αφορά τις απομακρύνσεις οργανικού υλικού, αζώτου και φωσφόρου) σε διάφορες αναλογίες άνθρακα προς άζωτο του τροφοδοτούμενου αποβλήτου ε) την εξέταση της σταθερότητας της βακτηριακής κοινότητας μέσω της τεχνικής της ηλεκροφόρησης σε πολυακρυλαμίδη σε διαβάθμιση αποδιατακτικών μέσων (DGGE) στ) τη σύνδεση συγκεκριμένων ομάδων μικροοργανισμών με την επεξεργασία του αποβλήτου. Η ποσοτικοποίηση του αριθμού γονιδίων πραγματοποιήθηκε με την τεχνική της Real-Time PCR. Συγκεκριμένα πραγματοποιήθηκε ο πολλαπλασιασμός των οξειδωτών αμμωνίας για τα αρχαία (AOA- ammonia oxidizing archaeal) και τα βακτήρια (AOB- ammonia Oxidizing Bacteria) (γονίδιο amoΑ), καθώς και ο πολλαπλασιασμός των γονιδίων απονιτροποίησης nirK, nirS, and nosZ.

Evaluation of DNA Colony Hybridization and Real-Time PCR for Detection of Vibrio parahaemolyticus and Vibrio vulnificus in Postharvest-Processed Oysters

2009 ◽  
Vol 72 (10) ◽  
pp. 2106-2109 ◽  
Author(s):  
JESSICA L. JONES ◽  
KATHY E. NOE ◽  
ROBIN BYARS ◽  
ANGELO DePAOLA
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
The United States ◽  
Most Probable Number ◽  
Probable Number ◽  
Colony Hybridization ◽  
Alkaline Peptone Water ◽  
Peptone Water

The applicability of real-time PCR was examined for detection of vibrios from postharvest-processed (PHP) oysters to allow for a more rapid assay and higher sample throughput than currently used. During June to October 2004, 68 PHP oyster samples were collected directly from PHP firms or from retail markets across the United States. PHP oysters were examined to determine the effectiveness of treatments in the reduction of vibrio levels and to compare the analytical methods utilized. The latter is the focus of the data presented here. Each sample was analyzed for Vibrio parahaemolyticus and V. vulnificus by using a 2-dilution, three-tube most-probable-number (MPN) and a 25-g presence/absence enrichment in alkaline peptone water. Following 6-h and overnight enrichment, aliquots from each MPN tube and the 25-g sample were streaked onto selective media and tested by real-time PCR. Colonies from the selective agar were confirmed as V. parahaemolyticus or V. vulnificus by DNA colony hybridization. DNA hybridization and real-time PCR results for each MPN tube and the 25-g enrichment at both time points were analyzed individually for each organism. The methods were in agreement for 857 (95%) of 901 and for 882 (98%) of 903 tubes for detection of V. parahaemolyticus and V. vulnificus, respectively. Overall, there was 96% agreement between real-time and DNA colony hybridization. The results obtained by real-time PCR were comparable to those from DNA colony hybridization, but analysis time was significantly reduced for the detection of vibrios in PHP-treated oysters.

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