Sains Malaysiana 43(12)(2014): 1965–1972

Development of the Double Digest Selective Label (DDSL) Typing Technique and Its Application to Staphylococcus aureus Epidemiology

(Pembangunan Label Terpilih Cernaan Berganda (DDSL) Teknik Penjenisan

dan Pemakaiannya dalam Epidemiologi Staphylococcus aureus)

V. TERLETSKIY¹, V. TYSHCHENKO¹, D.A. MYRZAKOZHA²*, O.O. ZHANSERKENOVA²,

Y.S. USSENBEKOV² & NURBEK L. ADAEV³

¹All-Russia Research Institute of Farm Animal Genetics and Breeding

Moscowskoye sh. 55a, 196625, St. Petersburg-Pushkin, Russia

²Kazakhstan National Agrarian University, Abaj str. 8, Almaty, Kazakhstan

³Chechen Research Institute of Agriculture, Lenin str. 1, Gikalo village, Groznij district,

366021 Russia

Diserahkan: 31 Disember 2012/Diterima: 13 Ogos 2014

ABSTRACT

Bacterial typing is a key technology in human and veterinary medicine, community health, consumer protection and in agricultural research. The importance of the development of epidemiological tracking tools is underlined by numerous outbreaks of diseases due to bacterial pathogens. Particularly important is tracing pathogen dissemination in ‘real time’ i.e. to use a fast typing technique to distinguish between clonally related (epidemic) strains and unrelated (sporadic) strains. The aim of the research was to develop a fast discriminatory molecular typing technique - double digest selective label (DDSL) for Staphylococcus aureus isolates and to compare typing data with that obtained by pulsed-field gel electrophoresis. In this new typing method, large DNA fragments are produced with a restriction enzyme commonly used for PFGE but are trimmed by a second enzyme to a size which can be separated on a conventional agarose gel within a short period of time. Selective labelling of a subset of the numerous restriction fragments gives a distinct banding pattern for each isolate. Discriminatory power obtained with DDSL calculated over two different sets was higher than that of pulsed-field gel electrophoresis. Clusters of identical isolates were further resolved to unique DDSL strains. Two combinations of restriction enzymes for DDSL technique has been proposed with approximately equal discriminatory power. It has been demonstrated that DDSL approach is a fast, discriminatory alternative to other typing techniques suitable for short-term epidemiological studies.

Keywords: DDSL; DNA; electrophoresis; epidemiology; fingerprinting; Staphylococcus aureus

 ABSTRAK

Penjenisan bakteria adalah teknologi utama dalam perubatan manusia dan haiwan, kesihatan masyarakat, perlindungan pengguna dan penyelidikan pertanian. Kepentingan pembangunan alat pengesanan epidemiologi digariskan oleh pelbagai wabak penyakit yang disebabkan oleh patogen bakteria. Adalah amat penting untuk mengesan penyebaran patogen secara ‘masa nyata’ iaitu untuk menggunakan teknik penjenisan pantas untuk membezakan antara strain berkaitan klon (wabak) dan strain tidak berkaitan (setempat-setempat). Kajian ini bertujuan untuk membangunkan teknik penjenisan diskriminasi molekul pantas- label terpilih cernaan berganda (DDSL) bagi pengasingan Staphylococcus aureus dan untuk membandingkan penjenisan data dengan yang diperoleh melalui elektroforesis jel medan denyut. Dalam kaedah penjenisan baru ini, cebisan DNA yang besar dihasilkan dengan enzim sekatan yang biasanya digunakan untuk PFGE tetapi dikemaskan oleh enzim kedua kepada saiz yang boleh dipisahkan dengan jel konvensional agarosa dalam tempoh masa yang singkat. Pelabelan terpilih sebahagian daripada cebisan sekatan yang banyak memberikan corak banding yang berlainan bagi setiap pencilan. Kuasa diskriminasi yang diperoleh dengan DDSL dikira menggunakan dua set yang berbeza adalah lebih tinggi daripada elektroforesis jel medan denyut. Kelompok pencilan seiras telah dileraikan kepada strain DDSL yang unik. Kombinasi dua sekatan enzim untuk teknik DDSL telah dicadangkan dengan kuasa diskriminasi yang hampir sama. Ia telah menunjukkan bahawa pendekatan DDSL adalah pantas dan diskriminasi alternatif kepada lain-lain teknik penjenisan yang sesuai untuk kajian epidemiologi jangka pendek.

Kata kunci: DDSL; DNA; elektroforesis; epidemiologi; pencapjarian; Staphylococcus aureus 

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*Pengarang untuk surat-menyurat; email: myrzakozha@yahoo.com