Development of a rapid and reliable TaqMan probe-based real-time PCR assay for the detection and enumeration of the multifaceted yeast Kluyveromyces marxianus in dairy products

Abstract

Kluyveromyces marxianus is a multifaceted yeast used in the dairy, biotechnological, and probiotic industries, and has been implicated in the spoilage of dairy products. Currently, K. marxianus detection involves laborious and time-consuming culture methods. To develop a rapid and reliable detection method for K. marxianus in pure cultures and dairy products, we designed a novel real-time PCR primer/probe set, KM55, which targets the internal transcribed spacer (ITS) rRNA gene. In the inclusivity and exclusivity tests, 11 K. marxianus strains tested positive, while 28 closely related microorganisms tested negative; this confirmed 100% sensitivity and specificity of the assay. Further, the KM55 set enabled a linear K. marxianus detection in the range of 10(0)-10(5) colony-forming units (CFU) per reaction in phosphate-buffered saline, 10% skim milk, and commercial yogurts, with no cross-reactivity with microorganisms of kefir origin. The enumeration of K. marxianus in kefir revealed 5.1 to 6.9 logCFU ml(-1) of kefir, depending on the fermentation time, temperature, and grain-milk ratio. K. marxianus was successfully detected in artificially contaminated yogurt samples within 2 h. These results suggest that this method could be an effective and sensitive presumptive screening tool for detecting and enumerating K marxianus in dairy products.