Incidence and Polymerase Chain Reaction Assay of Listeria monocytogenes from Raw Milk in Gyeongnam Province of Korea

2002 ◽  
Vol 65 (1) ◽  
pp. 111-115 ◽  
Author(s):  
KWANG-SOO HA ◽  
SEON-JA PARK ◽  
SOOK-JAE SEO ◽  
JUNG-HYUN PARK ◽  
DUCK-HWA CHUNG
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Raw Milk ◽  
Food Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
Milk Samples ◽  
Polymerase Chain ◽  
Pcr Assays

A total of 50 raw milk samples from Gyeongnam Province of Korea were examined for the incidence of Listeria monocytogenes between July 1998 and August 1998. L. monocytogenes isolated by biochemical test was confirmed by polymerase chain reaction (PCR) with two sets of primers designed from the invasion-associated protein (iap) gene. After standard PCR with external primers, the amplified DNA was confirmed by a second round of PCR with internal primers (nested PCR). Both the external and internal primers generated 468-bp and 287-bp products, respectively. Only one (G9 strain) of the three suspect samples that tested positive in biochemical tests for L. monocytogenes from 50 raw milk samples was also PCR positive. Following this procedure, PCR-positive G9 strain was confirmed by Southern blot using the 287-bp internal iap probe again. The detection limit of G9 strain by standard PCR assay was as few as 102 cells, equivalent to approximately 1 pg of L. monocytogenes DNA. These PCR assays may be useful for novel detection as well as rapid confirmation for L. monocytogenes from food samples and the field.

scholarly journals Streptococcus agalactiae mastitis of bovine detection by Polymerase Chain Reaction (PCR) test in AL-Diwanyia province

2013 ◽  
Vol 12 (2) ◽  
pp. 101
Author(s):  
Gh. K. A. Al-kuzaay ◽  
Q. H. Kshash
Keyword(s):  
Polymerase Chain Reaction ◽  
Streptococcus Agalactiae ◽  
Specific Primer ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Milk Samples ◽  
Bacteriological Testing ◽  
Polymerase Chain ◽  
Pcr Test

This study was conducted for exam 348 milk samples from (clinically mastitic and other healthy cows) in many areas in AL-Diwanyia province by using CMT and bacteriological testing , which appeared that (64.9%) as percentage of mastitis ( clinically 15.9% , subclinically 84.0% ) Streptococcus agalactiae mastitis 13.2% ( 26.6% clinically , 73.3 % subclinicaly) diagnose by PCR assay by using specific primer (16SrRNA). Streptococcus agalactiae (30 isolates) after classical methods applied for streptococcus agalactiae identification (86 isolates).

scholarly journals Application of a Multiplex Polymerase Chain Reaction Assay for the Simultaneous Confirmation of Listeria monocytogenes and Other Listeria Species in Turkey Sample Surveillance†

2002 ◽  
Vol 65 (5) ◽  
pp. 780-785 ◽  
Author(s):  
IRENE V. WESLEY ◽  
KAREN M. HARMON ◽  
JAMES S. DICKSON ◽  
ANN RAMOS SCHWARTZ
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Listeria Species ◽  
Multiplex Pcr Assay ◽  
Polymerase Chain

A multiplex polymerase chain reaction was developed to simultaneously identify Listeria monocytogenes and species of the genus Listeria. Two sets of primers were used, with the first amplifying a 938-bp region of the 16S rRNA gene that is highly conserved in all Listeria species and the second amplifying a 174-bp region of the listeriolysin (hlyA) gene of L. monocytogenes. Thus, isolates of Listeria spp. yield a single 938-bp product, whereas L. monocytogenes isolates yield both the 938-bp product and a 174-bp product. The specificity of the assay was verified with all six Listeria species and 11 serotypes of L. monocytogenes, as well as nonrelated bacteria. The multiplex PCR assay was used to determine the incidence of Listeria spp., especially L. monocytogenes, in mechanically separated turkey samples (n = 150 samples). L. monocytogenes strains were selected by using the University of Vermont two-step enrichment protocol and plating to selective Palcam agar. The multiplex PCR assay was used for verification of presumptive Listeria colonies. Approximately 38% of mechanically separated turkey samples (57 of 150) yielded L. monocytogenes; an additional 18% of these samples (27 of 150) harbored other Listeria spp. Fifty-one percent (29 of 57) of the L. monocytogenes isolates were of serogroup 1, 44% (25 of 57) were of serogroup 4, and 2% (1 of 57) were assigned to serogroups other than 1 and 4.

Combination of Immunomagnetic Separation and Polymerase Chain Reaction for the Simultaneous Detection of Listeria monocytogenes and Salmonella spp. in Food Samples

2001 ◽  
Vol 64 (11) ◽  
pp. 1744-1750 ◽  
Author(s):  
HSIEN-YEE HSIH ◽  
HAU-YANG TSEN
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Simultaneous Detection ◽  
Meat Products ◽  
Immunomagnetic Separation ◽  
Food Samples ◽  
Chain Reaction ◽  
Salmonella Spp ◽  
Pcr Method ◽  
Polymerase Chain

A method that combined the immunomagnetic separation (IMS) technique and the multiplex polymerase chain reaction (PCR) method (i.e., the IMS-mPCR method) was developed for simultaneous detection of Listeria monocytogenes and Salmonella spp. in food samples. When only the multiplex PCR method was used, it was found that if cell numbers of each of the two target organisms (L. monocytogenes and Salmonella spp.) were above the detection limit, but differed by more than 2 logs—e.g., n × 107 to n × 104 or n × 106 to n × 103—the organism presenting the lower numbers might go undetected. Following the enrichment step with universal preenrichment (UP) broth, if an IMS method using equal quantities of anti-Listeria and anti-Salmonella immunomagnetic beads was performed prior to PCR, both pathogens could be detected unambiguously. Such results could be obtained for target organisms in food samples, such as milk, dairy, and meat products, if similar enrichment and IMS steps were performed prior to PCR.

Multiplex polymerase chain reaction as a mastitis screening test for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis in bulk milk samples

2003 ◽  
Vol 70 (2) ◽  
pp. 149-155 ◽  
Author(s):  
Patchara Phuektes ◽  
Glenn F Browning ◽  
Garry Anderson ◽  
Peter D Mansell
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Streptococcus Dysgalactiae ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Milk Samples ◽  
Streptococcus Uberis ◽  
Bulk Milk ◽  
Polymerase Chain ◽  
Total Bacteria

Effective diagnostic tools for screening herds for mastitis pathogens are important in development and monitoring of mastitis control programmes. A multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis was used in preliminary studies to assess its applicability as an alternative method for monitoring mastitis caused by these organisms at the herd level. PCR was used to detect the presence of these organisms in bulk milk samples. Correlations with bulk milk somatic cell counts (BMCC), total bacteria counts and thermoduric bacteria counts were evaluated. A total of 176 bulk milk samples were collected from 42 herds on five consecutive occasions at approx. 10-d intervals. Str. uberis was the most common organism in these bulk milk samples. There was no relationship between presence of either Staph. aureus, Str. dysgalactiae or Str. uberis and BMCC, total bacteria counts or thermoduric bacteria counts. However, presence of Str. agalactiae was associated with high BMCC and total bacteria counts. The results of this study show that regular analysis of bulk milk using this multiplex PCR assay may be a useful tool for monitoring herd status with respect to Str. agalactiae, but is of less value for monitoring occurrence of Staph. aureus, Str. dysgalactiae and Str. uberis. Further investigations are needed to clarify the relationship between positive PCR results and the prevalence of infected cows in the herd.

scholarly journals Daignosis of Staphylococcus aureus mastitis in bovine in Al-Najaf province by using Polymerase chain reaction (PCR)

2013 ◽  
Vol 12 (2) ◽  
pp. 81
Author(s):  
N. A. Al- Anbagi
Keyword(s):  
Staphylococcus Aureus ◽  
Polymerase Chain Reaction ◽  
Subclinical Mastitis ◽  
Molecular Method ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Milk Samples ◽  
Significant Incidence ◽  
Left Posterior ◽  
Polymerase Chain

This study was conducted to collect 388 milk samples from cows at different villages and townships in Al-Najaf province to examine about Staphylococcus aureus mastitis .CMT was used for subclinical mastitis screening ,212(54.6%) milk samples were mastitic .The molecular method (PCR assay) was used to detected the presence (glpF) gene in classically diagnosed S.aureus, which appeared that 38(92.6%) S.aureus mastitis as 13(32.5%) clinical and 25(14.5%) subclinical mastitis .There was high significant incidence of Staphylococcus aureus mastitis in left posterior udder quarter rather than others quarters

Polymerase Chain Reaction Assay Coupled with Fluorescence Detection on Microwell Plates for Listeria monocytogenes in Foods

1995 ◽  
Vol 58 (6) ◽  
pp. 614-620 ◽  
Author(s):  
RAÚL J. CANO ◽  
DAWN M. NORTON ◽  
ALMA E. INZUNZA ◽  
JOSÉ GIL SÁNCHEZ ◽  
CHRISTIAN OSTE
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Fluorescence Detection ◽  
Polymerase Chain Reaction Assay ◽  
Food Samples ◽  
Fluorescence Measurement ◽  
Chain Reaction ◽  
Predictive Values ◽  
Dairy Food ◽  
Polymerase Chain

This study evaluates a polymerase chain-reaction assay coupled with fluorescence detection (PCR-FD) in microwell plates for Listeria monocytogenes in dairy and food samples. Guanidinium thiocyanate/silica–extracted cultures and milk and dairy-food samples were used as templates for a PCR assay in microwell plates, with a primer pair that amplifies a 221 bp segment of the internal transcribed spacer (ITS), in the presence of 0.5 μg/ml of ethidium bromide. Fluorescence of the PCR products was measured with a CytoFluor 2300 fluorescence measurement system (Millipore Corporation, Bedford, MA). The lowest level of detection of the assay was 10 to 100 CFU. A total of 326 food samples were tested, both by culture and by PCR-FD. The overall sensitivity of the PCR-FD assay was 95.2% and the specificity was 89.9%. Positive and negative predictive values were 74.8% and 99.4%, respectively. Based on the results obtained in this study it appears that the PCR-FD assay described here can be useful for screening a large number of milk and dairy-food samples for contamination by Listeria monocytogenes.

Detection of Thermophilic Campylobacter spp. in Blood-Free Enriched Samples of Inoculated Foods by the Polymerase Chain Reaction

2000 ◽  
Vol 63 (3) ◽  
pp. 299-303 ◽  
Author(s):  
RICHARD L. THUNBERG ◽  
TONY T. TRAN ◽  
MARK O. WALDERHAUG
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Raw Milk ◽  
Extraction Techniques ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Enrichment Broth ◽  
Thermophilic Campylobacter ◽  
Polymerase Chain ◽  
Campylobacter Spp

The detection of thermophilic Campylobacter spp., as represented by Campylobacter jejuni, by the polymerase chain reaction (PCR) was investigated and compared with the selective agar isolation (SAI) method. Stationary-phase cultures of C. jejuni were inoculated into either blood-free enrichment broth (BFEB) or BFEB that contained 10% broccoli, crabmeat, mushroom, raw milk, and raw oyster rinses. Following a 48-h enrichment period, aliquots of food test portions were removed for simultaneous analysis by PCR and SAI. It was determined that the presence of charcoal and iron in the enrichment broth interfered with the PCR assay. Therefore, three DNA extraction techniques were developed and evaluated using a 16S rRNA primer pair in the PCR assay. The 50% end point (DL50) values (determined upon six initial C. jejuni spiking levels) were used to assess the frequency of isolation utilizing PCR versus SAI for the detection of this organism in the enrichment matrices. There were virtually no differences in detection of C. jejuni among enriched samples analyzed by PCR and SAI. Mean DL50 values (n = 3) for plain BFEB, broccoli, crabmeat, mushroom, raw milk, and raw oyster were, respectively, 0.02 (PCR) versus 0.01 (SAI), 0.01 versus 0.06, 0.07 versus 0.04, 0.03 versus 0.08, 0.01 versus 0.01, and 0.01 versus 0.01 CFU/5 g food. Significant variability in the detection limit of C. jejuni by PCR in the food enrichments was observed among DNA extraction techniques. Using 48-h enrichment cultures followed by PCR analysis could save 1 day of the time required for the presumptive identification of C. jejuni in suspected foods.

Polymerase Chain Reaction Detection of Listeria monocytogenes on Frankfurters Using Oligonucleotide Primers Targeting the Genes Encoding Internalin AB

2003 ◽  
Vol 66 (2) ◽  
pp. 237-241 ◽  
Author(s):  
YONG SOO JUNG ◽  
JOSEPH F. FRANK ◽  
ROBERT E. BRACKETT ◽  
JINRU CHEN
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Meat Products ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Oligonucleotide Primers ◽  
Pcr Product ◽  
Genes Encoding ◽  
Pure Cell ◽  
Polymerase Chain

A polymerase chain reaction (PCR) assay targeting the genes encoding internalin AB (inlAB) was developed for detecting Listeria monocytogenes in pure cell cultures and on artificially contaminated frankfurters. Four sets of oligonucleotide primers were evaluated. The set targeting a 902-bp region of the inlAB gene was the most specific. This PCR product was detected in 51 L. monocytogenes strains belonging to four different serogroups (1/2a, 1/2b, 1/2c, and 4b). In contrast, the PCR product was not detected in other Listeria spp. (Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri, or Listeria grayi) or in gram-positive, non-Listeria bacteria, indicating that the primer set was highly specific for L. monocytogenes. The detection limit of the PCR assay was 105 CFU per ml of pure cell culture. However, the assay could detect as few as 101 CFU of L. monocytogenes in 25 g of frankfurter with 16 h of enrichment in modified Listeria enrichment broth at 30°C. The total assay time including enrichment was approximately 24 h. These results suggest that the PCR assay can be used to rapidly detect L. monocytogenes on frankfurters and possibly other types of ready-to-eat meat products.

scholarly journals Characterization of Campylobacter jejuni An Important Zoonotic Food Borne Pathogen from Mastitis Milk and Raw Milk Samples

2021 ◽  
Author(s):  
Sumedha Bobade ◽  
K. Vijayarani ◽  
K.G. Tirumurugaan ◽  
A. Thangavelu ◽  
S. Vairamuthu
Keyword(s):  
Polymerase Chain Reaction ◽  
Campylobacter Jejuni ◽  
Morphological Characteristics ◽  
Raw Milk ◽  
Phenotypic Characterization ◽  
Chain Reaction ◽  
Milk Samples ◽  
Food Borne ◽  
Polymerase Chain ◽  
Pcr Targeting

Background: Campylobacter has emerged as an important zoonotic food borne pathogen of human and animals worldwide. Campylobacter is one of the most common bacterial enteropathogens of food borne origin in industrialized countries with C. jejuni being the most common species followed by C. coli. There are very few cases reported from mastitis therefore this study was aimed to determine the incidence of Campylobacter jejuni from from mastitis milk and raw milk samples. Methods: Total of 72 milk samples comprising mastitis milk (20) and raw milk (52) were collected. The samples were subjected to cultural examination, biochemical as well as molecular identification. The isolates were further subjected to phenotypic characterization by biochemical test and genotypic characterization by Polymerase Chain Reaction. The isolates were subjected to PCR targeting hip O and MAP A genes. Result: The 52 samples showed growth on modified Blood Free Charcoal Cefoperazone Deoxycholate agar media and 18 (34.61%) samples showed typical morphological characteristics. The result revealed that 10 (19.23%) isolates were positive by phenotypic characteristic and 7(70%) by Polymerase chain reaction for C. jejuni. The outcome result showed that importance of Campylobacter jejuni in cattle, especially raw milk and milk from mastitis cows, as a potential source for transmission of Campylobacteriosis in human and dairy farm environment. This can cause acute bacterial gastroenteritis in humans and associated with foodborn infection, food safety and a serious public health threat.

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