Rapid enumeration of Listeria monocytogenes in milk using competitive PCR.
Competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Listeria monocytogenes in milk. Sterile milk was artificially inoculated with L. monocytogenes and DNA was extracted using guanidine thiocyanate/phenol/chloroform, followed by PCR. Several primers for L. monocytogenes hlyA gene were tested for specific detection and DG69/DG74 primer set was selected. The primer set produced a 636-bp band from L. monocytogenes, but no band appeared from the other six Listeria spp. tested. A detection limit was as few as 10(3) colony-forming unit (cfu) per 0.5 ml of milk with this primer set. When the samples were cultured at 25 degrees C for 15 h in a TSBY medium, even a single bacterium could be detected with this primer set by PCR. For the cPCR, hlyA gene segment was cloned in pGem-4Z vector and was modified to produce competitor DNA. The competitor DNA has the same primer binding sites and sequences as the target DNA except EcoRI site. Known amount of competitor DNA was coamplified with L. monocytogenes total DNA isolated from artificially inoculated milk. The target DNA and competitor DNA were distinguished by EcoRI digestion after cPCR. The cell number determined by cPCR was approximately equal to the colony-forming unit from conventional plate counting method. For the whole procedure, it took only 5 h.
Choi
Weon Sang
WS
Department of Biotechnology, College of Natural Sciences, Dongguk University, Gyeongju 780-714, Republic of Korea. weonsang@mail.dongguk.ac.kr
Hong
Chong-Hae
CH
D000818:Animals; D001427:Bacterial Toxins; D015169:Colony Count, Microbial / Q000379:methods; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D005516:Food Microbiology; D006360:Heat-Shock Proteins / Q000235:genetics; D006460:Hemolysin Proteins; D008089:Listeria monocytogenes / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
D001427:Bacterial Toxins; D017931:DNA Primers; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; C038705:hlyA protein, Listeria monocytogenes