scholarly journals Development of a Fluorogenic Probe-Based PCR Assay for Detection of Bacillus cereus in Nonfat Dry Milk

2000 ◽  
Vol 66 (4) ◽  
pp. 1453-1459 ◽  
Author(s):  
Young-Rok Kim ◽  
John Czajka ◽  
Carl A. Batt
Keyword(s):  
Bacillus Cereus ◽  
Positive Sequence ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Probable Number ◽  
Fluorogenic Probe ◽  
Hemolysin Gene ◽  
Dry Milk ◽  
Fluorogenic Probes ◽  
Nonfat Dry Milk

ABSTRACT A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5′-to-3′ exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereusstrains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negativeB. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated withB. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.

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 Evaluation of Two Direct Plating Methods Using Nonradioactive Probes for Enumeration of Vibrio parahaemolyticus in Oysters

2001 ◽  
Vol 67 (2) ◽  
pp. 721-724 ◽  
Author(s):  
J. A. Gooch ◽  
A. DePaola ◽  
C. A. Kaysner ◽  
D. L. Marshall
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Most Probable Number ◽  
Mobile Bay ◽  
Probable Number ◽  
Direct Plating ◽  
Hemolysin Gene ◽  
Highly Correlated ◽  
Species Specific ◽  
Labeled Probe ◽  
Zero Time

ABSTRACT Oysters (Crassostrea virginica) were collected monthly from May 1998 to April 1999 from Mobile Bay, Ala., and analyzed to determine Vibrio parahaemolyticus densities at zero time and after 5, 10, and 24 h of postharvest storage at 26°C. After 24 h of storage at 26°C, oysters were transferred to a refrigerator at 3°C and then analyzed 14 to 17 days later. TheV. parahaemolyticus numbers were determined by the most-probable-number procedure using alkaline phosphatase-labeled DNA probe VPAP, which targets the species-specific thermolabile hemolysin gene (tlh), to identify suspect isolates (MPN-VPAP procedure). Two direct plating methods, one using a VPAP probe (Direct-VPAP) and one using a digoxigenin-labeled probe (Direct-VPDig) to identify suspect colonies, were compared to the MPN-VPAP procedure. The results of the Direct-VPAP and Direct-VPDig techniques were highly correlated (r = 0.91), as were the results of the Direct-VPAP and MPN-VPAP procedures (r = 0.91). The correlation between the Direct-VPDig and MPN-VPAP results was 0.85. The two direct plating methods in which nonradioactive DNA probes were used were equivalent to the MPN-VPAP procedure for identification of totalV. parahaemolyticus, and they were more rapid and less labor-intensive.

scholarly journals Prevalence of Bacillus cereus s.l. in ultra-high temperature chocolate milk from selected milk manufacturers in Malaysia

Food Research ◽  
2020 ◽  
Vol 4 (4) ◽  
pp. 982-990
Author(s):  
Ubong Anyi ◽  
C.Y. New ◽  
L.C. Chai ◽  
Y.Y. Loo ◽  
Nor Khaizura M.A.R. ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Foodborne Pathogen ◽  
Most Probable Number ◽  
Contamination Level ◽  
Chocolate Milk ◽  
Probable Number ◽  
Potential Source ◽  
Milk Contamination ◽  
Bacillus Spp ◽  
Polymerase Chain

Bacillus cereus is a major foodborne pathogen of great concern to the dairy industry owing to its resilient spores as well as the adverse effect of its toxins. At present, there is no informational study available to solve or pinpoint the UHT chocolate milk contamination issue in Malaysia. This work aimed to investigate the prevalence and contamination level of B. cereus s.l. in UHT chocolate milk and to suggest the appropriate solution for the issue. In the present study, B. cereus s.l. prevalence and contamination level in individually packed UHT chocolate milk from processing factories was evaluated. The prevalence and concentration of B. cereus s.l. were determined via MPN-PCR (Most Probable Number-Polymerase Chain Reaction) assay. Results showed that 31.11% from 220 of UHT chocolate milk tested contained Bacillus spp.; of this Bacillus spp. positive samples, 24.30% were also positive for B. cereus s.l. with concentration ranging from less than 3 to more than 1100 MPN/mL. Findings from this study highlighted the possibility of UHT chocolate milk as a potential source of B. cereus s.l. infection. Therefore, findings emphasized the needs to revise, monitor and improve UHT sterilization process to reduce infection risk. Furthermore, it is also essential to maintain the hygiene to minimize initial microbial load and contamination of UHT chocolate milk, beginning from production to retail.

scholarly journals Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples

2001 ◽  
Vol 67 (4) ◽  
pp. 1613-1618 ◽  
Author(s):  
Line Fredslund ◽  
Flemming Ekelund ◽  
Carsten Suhr Jacobsen ◽  
Kaare Johnsen
Keyword(s):  
Dna Sequences ◽  
Sequence Data ◽  
Small Subunit ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Heterotrophic Flagellates ◽  
Small Subunit Rrna ◽  
Pcr Assay ◽  
Probable Number ◽  
Soil Protozoa

ABSTRACT This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.

Effects of Dry Storage and Resubmersion of Oysters on Total Vibrio vulnificus and Total and Pathogenic (tdh+/trh+) Vibrio parahaemolyticus Levels

2015 ◽  
Vol 78 (8) ◽  
pp. 1574-1580 ◽  
Author(s):  
THOMAS P. KINSEY ◽  
KERI A. LYDON ◽  
JOHN C. BOWERS ◽  
JESSICA L. JONES
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Storage Time ◽  
Vibrio Vulnificus ◽  
Ambient Air ◽  
Most Probable Number ◽  
Probable Number ◽  
Ambient Temperatures ◽  
Dry Storage ◽  
Hemolysin Gene ◽  
Thermostable Direct Hemolysin

Vibrio vulnificus (Vv) and Vibrio parahaemolyticus (Vp) are the two leading causes of bacterial illnesses associated with raw shellfish consumption. Levels of these pathogens in oysters can increase during routine antifouling aquaculture practices involving dry storage in ambient air conditions. After storage, common practice is to resubmerge these stored oysters to reduce elevated Vv and Vp levels, but evidence proving the effectiveness of this practice is lacking. This study examined the changes in Vv and in total and pathogenic (thermostable direct hemolysin gene and the tdh-related hemolysin gene, tdh+ and trh+) Vp levels in oysters after 5 or 24 h of dry storage (28 to 32°C), followed by resubmersion (27 to 32°C) for 14 days. For each trial, replicate oyster samples were collected at initial harvest, after dry storage, after 7 days, and after 14 days of resubmersion. Oysters not subjected to dry storage were collected and analyzed to determine natural undisturbed vibrio levels (background control). Vibrio levels were measured using a most-probable-number enrichment followed by real-time PCR. After storage, vibrio levels (excluding tdh+ and trh+ Vp during 5-h storage) increased significantly (P < 0.001) from initial levels. After 7 days of resubmersion, Vv and total Vp levels (excluding total Vp in oysters stored for 5 h) were not significantly different (P > 0.1) from levels in background oysters. Vv and total and pathogenic Vp levels were not significantly different (P > 0.1) from levels in background oysters after 14 days of resubmersion, regardless of dry storage time. These data demonstrate that oyster resubmersion after dry storage at elevated ambient temperatures allows vibrio levels to return to those of background control samples. These results can be used to help minimize the risk of Vv and Vp illnesses and to inform the oyster industry on the effectiveness of routine storing and resubmerging of aquaculture oysters.

Incidence and Growth Potential of Bacillus cereus in Ready-to-Serve Foods

1991 ◽  
Vol 54 (5) ◽  
pp. 372-374 ◽  
Author(s):  
STANLEY M. HARMON ◽  
DONALD A. KAUTTER
Keyword(s):  
Bacillus Cereus ◽  
Infant Formula ◽  
Growth Potential ◽  
Powdered Infant Formula ◽  
Powdered Milk ◽  
Nonfat Milk ◽  
Dry Milk ◽  
Nonfat Dry Milk

To simulate temperature abuse, 106 test portions of ready-to-serve moist foods, 12 test portions of rehydrated powdered infant formula, and 18 test portions of nonfat dry milk were incubated for 20 and 24 h at 26°C, and then examined for Bacillus cereus. Of the ready-to-serve moist foods, 88 of 106 were positive for B. cereus at levels ranging from 0.25 to 8.5 × 106/g after 20 h of incubation and from 0.1 to 58 × 106/g after 24 h. All of the powdered milk and 12 of the 15 units of infant formula, representing five brands, were positive, with counts ranging from 0.15 to 5.0 × 106/g in 20 h and 5.0 to 49 × 106 after 24 h. B. cereus counts in the powdered products were low, ranging from 0.09/g for one of two soy-based products to an average of 0.29/g for milk-based products. However, these levels were sufficient to initiate growth of B. cereus in almost every 2-oz serving. Similar results were obtained for rehydrated nonfat milk, with initial B. cereus counts ranging from 0.29 to 1.5/g; at 26°C the counts averaged 3.3 × 107 after 20 h and 5.5 × 107 after 24 h. Counts ranged from 2.0 × 104 to 1.1 × 105 after 9 h in milk and were in excess of 106/g after 10.5 h.

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.

Detection of Escherichia albertii in retail oysters

2021 ◽  
Author(s):  
Sakura Arai ◽  
Satoko Yamaya ◽  
Kayoko Ohtsuka ◽  
Noriko Konishi ◽  
Hiromi Obata ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Pacific Oyster ◽  
Genetic Characterization ◽  
Foodborne Pathogen ◽  
Most Probable Number ◽  
Macconkey Agar ◽  
Pcr Assay ◽  
Probable Number ◽  
Pacific Oysters ◽  
Rock Oyster

Escherichia albertii  is an emerging foodborne pathogen. Owing to its distribution in river water,  it is important to determine the presence of  E. albertii  in aquaculture-related foods. In this study, we investigated the distribution of  E. albertii  in retail oyster samples.  A total of  427 raw oyster samples (385 Pacific oysters, and 42 Japanese rock oysters) were enriched in  modified Escherichia coli  broth (mEC) or mEC supplemented with novobiocin (NmEC) at 42 °C. The cultures were used for  E. albertii -specific nested PCR assay, as well as for  E. albertii  isolation using  deoxycholate hydrogen sulfide lactose agar  (DHL), DHL supplemented with rhamnose and xylose (RX-DHL), and MacConkey agar supplemented with rhamnose and xylose (RX-MAC). The population of  E. albertii  in nested PCR-positive samples was  determined using the  most probable number  (MPN) method.  E. albertii  isolates were subjected to biochemical and genetic characterization.  E. albertii   was detected in 5 of 315 (1.6%) Pacific oyster samples  (one piece each), 2 of 70 (2.9 %)  Pacific oyster samples  (25 g each), and 2 of 42 (4.8 %) Japanese rock oyster samples  procured from four geographically distant regions. A total of 64  E. albertii  strains were isolated from eight of the nine nested PCR assay-positive oyster samples, and  the MPN value was under the detection limit (&lt; 3 MPN/10 g).  A specific season or month for detecting  E. albertii  was not observed in this study, suggesting that the pathogen is present in seawater.   All the  E. albertii  isolates, except one, were positive for the virulence factor  eae,  indicating that these isolates have  the potential to infect humans.

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