A 23S rDNA-Targeted Polymerase Chain Reaction–Based System for Detection of Staphylococcus aureus in Meat Starter Cultures and Dairy Products

1999 ◽  
Vol 62 (10) ◽  
pp. 1150-1156 ◽  
Author(s):  
JOACHIM A. STRAUB ◽  
CHRISTIAN HERTEL ◽  
WALTER P. HAMMES
Keyword(s):  
Staphylococcus Aureus ◽  
Polymerase Chain Reaction ◽  
Skim Milk ◽  
Starter Cultures ◽  
23S Rrna ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Selective Enrichment ◽  
Polymerase Chain

A polymerase chain reaction–based system for detection of Staphylococcus aureus was developed. The system consisted of the following components: (i) selective enrichment, (ii) DNA isolation, (iii) amplification of DNA with primers targeted against the 23S rRNA gene, and (iv) evaluation of the specificity of the polymerase chain reaction by Southern hybridization and nested polymerase chain reaction. The method achieved a high degree of sensitivity and unambiguity as required for the detection of contaminants in food starter preparations. The method permitted detection of Staphylococcus aureus in preparations of meat starter cultures containing Staphylococcus carnosus either alone or in combination with lactobacilli, pediococci, and/or Kocuria varians. Detection limits were sufficiently low to show within 12 h the presence of 100 CFU of S. aureus in starter preparations containing 1010 CFU of S. carnosus. The system was also applied to dried skim milk and cream. For detection without selective enrichment, a protocol was developed and permitted detection of 120 CFU of S. aureus in 1 ml of cream within 6 h. With nested polymerase chain reaction, the detection limit was decreased by one order of magnitude.

scholarly journals Improved Multiplex Polymerase Chain Reaction for Rapid Staphylococcus Aureus Detection in Meat and Milk Matrices

2016 ◽  
Vol 15 (1) ◽  
pp. 65-76
Author(s):  
Zuzana Šramková ◽  
Barbora Vidová ◽  
Andrej Godány
Keyword(s):  
Staphylococcus Aureus ◽  
Polymerase Chain Reaction ◽  
Food Poisoning ◽  
Detection Sensitivity ◽  
23S Rrna ◽  
Rrna Gene ◽  
Chain Reaction ◽  
23S Rrna Gene ◽  
Polymerase Chain ◽  
Toxic Shock Syndrome Toxin

Abstract Staphylococcal food poisoning represents one of the most frequently occurring intoxications, caused by staphylococcal enterotoxins (SE-s) and staphylococcal enterotoxin-like proteins (SEl-s). Therefore, there is a need for rapid, sensitive and specific detection method for this human pathogen and its toxin genes in food matrices. The present work is focused on Staphylococcus aureus detection by a nonaplex polymerase chain reaction, which targets the 23S rRNA gene for identification of S. aureus at the species level, genes for classical SE-s (SEA, SEC, SED), new SE-s (SEH, SEI), SEl-s (SEK, SEL) and tsst-1 gene (toxic shock syndrome toxin). Primers were properly designed to avoid undesirable interactions and to create a reliably identifiable profile of amplicons when visualized in agarose gel. According to obtained results, this approach is able to reach the detection sensitivity of 12 colony forming units from milk and meat matrices without prior culturing and DNA extraction.

Development of a Multiplex Polymerase Chain Reaction Assay for Detection and Differentiation of Staphylococcus aureus in Dairy Products†

2001 ◽  
Vol 64 (5) ◽  
pp. 664-668 ◽  
Author(s):  
SUDHIR TAMARAPU ◽  
JOHN L. McKILLIP ◽  
MARYANNE DRAKE
Keyword(s):  
Staphylococcus Aureus ◽  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Dairy Products ◽  
Multiplex Polymerase Chain Reaction ◽  
Skim Milk ◽  
Cheddar Cheese ◽  
Polymorphism Analysis ◽  
Chain Reaction ◽  
Polymerase Chain

A multiplex polymerase chain reaction (PCR) assay was developed for the detection and differentiation of enterotoxigenic Staphylococcus aureus in dairy products. A solvent extraction procedure was successfully modified for extraction of S. aureus DNA from 10 ml of artificially contaminated skim milk or 20 g cheddar cheese. Primers targeting the enterotoxin C gene (entC) and thermostable nuclease gene (nuc) were used in the multiplex PCR. PCR products were confirmed using restriction fragment length polymorphism analysis. DNA was consistently quantified and amplified by uniplex PCR from 10 CFU/ml of S. aureus in skim milk or 10 CFU/20 g cheddar cheese. The sensitivity of the multiplex PCR was 100 CFU/ml of skim milk or 100 CFU/20 g cheddar cheese. The developed methodology allows presumptive identification and differentiation of enterotoxigenic S. aureus in less than 6 h.

scholarly journals Identifikasi Molekuler Bakteri Staphylococcus sp. dan Staphylococcus aureus Penyebab Mastitis Subklinis pada Ternak Kambing Perah

2021 ◽  
Vol 39 (2) ◽  
pp. 151
Author(s):  
Clara Ajeng Artdita ◽  
Fatkhanuddin Aziz ◽  
Nurulia Hidayah ◽  
Achmad Fauzi ◽  
Triastuti Septi Wulandari ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Polymerase Chain Reaction ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
23S Rrna ◽  
Chain Reaction ◽  
Methicillin Resistant ◽  
Polymerase Chain

Mastitis merupakan radang pada glandula mammae (ambing) ternak perah. Mastitis tipe subklinis sering dikaitkan pada kejadian mastitis di peternakan ruminansia kecil seperti kambing perah (kambing Peranakan Etawah, Saanen, dan Sapera). Patogen utama yang berperan dalam kejadian mastitis ini adalah genus Staphylococcus. Tujuan penelitian adalah untuk melakukan identifikasi bakteri Staphylococcus sp. dan Staphylococcus aureus sebagai penyebab mastitis subklinis pada kambing perah dengan menggunakan metode polymerase  chain reaction (PCR). Tahapan metode yang dilakukan adalah ekstraksi DNA dengan teknik spin-collumn system terhadap 26 isolat bakteri yang telah dilakukan uji biokimia sebelumnya dan amplifikasi gen spesifik 23s rRNA Staphylococcus sp. dan Staphylococcus aureus, serta methicillin resistant Staphylococcus aureus (MRSA), dilanjutkan dengan visualisasi menggunakan UV-transluminator. Hasil menunjukkan bahwa sebanyak 12 isolat sampel teridentifikasi Staphylococcus sp. dan 1 diantaranya teridentifikasi Staphylococcus aureus. Isolat yang teridentifikasi Staphylococcus aureus bukan termasuk MRSA. 

Proteolytic activity of lactococcal strains from water-buffalo Mozzarella starter cultures

1998 ◽  
Vol 65 (1) ◽  
pp. 109-118 ◽  
Author(s):  
GIANLUIGI MAURIELLO ◽  
GIANCARLO MOSCHETTI ◽  
GIUSEPPE BLAIOTTA ◽  
FRANCESCO VILLANI ◽  
SALVATORE COPPOLA
Keyword(s):  
Polymerase Chain Reaction ◽  
Proteolytic Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Skim Milk ◽  
Starter Cultures ◽  
Chain Reaction ◽  
Heat Treated ◽  
Genes Encoding ◽  
Specific Amplification ◽  
Polymerase Chain

Growth and proteolytic activity of Lactococcus lactis strains were investigated in milks subjected to different heat treatments. Only 13·7% of the strains were of proteolytic phenotype. Proteinase-positive (Prt+) strains exhibited a proteolytic activity that increased when the level of heat treatment was reduced and grew to a higher level than proteinase-negative (Prt−) strains in low-heat-treated milk. When grown in autoclaved reconstituted skim milk, both Prt− and Prt+ strains had the same specific growth rate. Total DNA from four Prt+ and eleven Prt− strains was extracted and used for polymerase chain reaction amplification of fragments belonging to genes encoding for cell wall proteinase. Specific amplification products were displayed by all the Prt+ strains, and by the Prt− strain Lc. lactis subsp. lactis NWC 125. Reverse transcriptase-polymerase chain reaction was performed to detect the transcript of the proteinase genes. In addition to the Prt+ strains, the experiment also detected the specific transcript in Lc. lactis subsp. lactis NWC 125, suggesting that other structural or functional deficiencies occurred in this strain.

scholarly journals DETEKSI GEN tst ISOLAT Staphylococcus aureus MELALUI AMPLIFIKASI 23S rRNA ASAL SUSU KAMBING DAN SAPI PERAH

2014 ◽  
Vol 8 (1) ◽  
Author(s):  
Budi Prasetyo ◽  
Elizabeth Novi Kusumaningrum
Keyword(s):  
Staphylococcus Aureus ◽  
Polymerase Chain Reaction ◽  
Deoxyribonucleic Acid ◽  
23S Rrna ◽  
Chain Reaction ◽  
Polymerase Chain

Penelitian ini bertujuan mendeteksi gen tst isolat Staphylococcus aureus (S. aureus) susu sapi perah dan susu kambing melalui amplifikasi gen 23S rRNA sebagai langkah awal pencegahan beberapa kasus keracunan susu. Metode penelitian meliputi reidentifikasi bakteri, preparasi deoxyribonucleic acid (DNA), amplifikasi gen 23S rRNA, amplifikasi gen tst, dan pengurutan DNA. Hasil reidentifikasi bakteri melalui pewarnaan Gram, uji katalase, uji koagulase, uji fermentasi MSA, uji VJA, dan uji VP adalah positif bakteri S. aureus. Hasil amplifikasi gen tst terhadap 3 isolat S. aureus menunjukkan hanya 2 isolat memberikan hasil positif. Hasil positif tersebut ditandai dengan munculnya fragmen DNA yang memiliki panjang spesifik (350 bp) sesuai dengan produk polymerase chain reaction (PCR) dari referensi dan database GeneBank. Disimpulkan bahwa dengan menggunakan metode PCR dapat terdeteksi adanya gen tst pada isolat S. aureus asal susu sapi perah dan susu kambing dari Bogor.

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