Sains Malaysiana 39(1)(2010): 57–63

 

Detection of Shiga Toxin 1 and 2 (stx1 and stx2) Genes in Escherichia coli O157:H7 Isolated from Retail Beef in Malaysia by Multiplex

Polymerase Chain Reaction (PCR)

(Pengesanan Gen Toksin Shiga 1 dan 2 (stx1 dan stx2) dalam Escherichia coli O157:H7 

yang dipencilkan daripada Daging Lembu Runcit di Malaysia dengan

Menggunakan Tindak Balas Rantaian Polimerase Multipleks)

 

A.M. Sahilah*

School of Chemical Sciences and Food Technology

Faculty of Science and Technology, Universiti Kebangsaan Malaysia

43600 UKM Bangi, Selangor D.E., Malaysia

 

H. Nor' Aishah & I. Noraida

Institute Biological Sciences, Faculty of Science

University of Malaya, 50306 Kuala Lumpur, Malaysia

 

A. Ahmad Azuhairi

Department of Community Health

Faculty of Medicine and Health Science, Universiti Putra Malaysia

43300 Serdang Selangor D.E., Malaysia

 

Received: 12 March 2009 / Accepted: 27 April 2009

 

ABSTRACT

Twenty (n=20) beef isolates of Escherichia coli O157:H7 were examined for the detection of Shiga- toxin 1 and 2 (stx1 and stx2) genes by multiplex polymerase chain reaction (PCR) and characterized using Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) fingerprinting. All isolates were obtained from the laboratory of Food Science and Biotechnology, University Putra Malaysia, Serdang, Selangor. In the detection of stx1 and stx2 genes, 14 of isolates (14/20) were positive to stx1 and stx2. 5 isolates (5/20) were positive to stx1 and 1 isolate (1/20) was negative by either of stx1 or stx2 genes. Using RAPD-PCR analysis, two oligonucleotides were chosen because they yielded clearly and reproducible band. There were OPAR8 (5'-TGGGGCTGTC-3') and OPAR20 (5'-ACGGCAAGGA-3'). Subsequently, all 20 isolates of E.coli O157:H7 were subtyped using OPAR8 and OPAR20. Primer OPAR8 produced 8 RAPD-PCR fingerprinting namely P1 to P11. Whereas, OPAR20 produced 16 RAPD-PCR fingerprinting of Q1-Q18. Combination of two primers was analyzed using Unweighted Pair Group Method with Arithmetic mean (UPGMA). Dendogram performed from cluster analysis showed that all the 20 isolates of E.coli O157:H7 differentiated into 20 individual isolates which may suggest the high level of local geographical genetic variation.

 

Keywords: Escherichia coli O157:H7; multiplex PCR; retail beef; Stx1 and stx2 genes

 

 

ABSTRAK

 

Dua puluh (n=20) isolat Escherichia coli O157:H7 yang diasingkan daripada daging lembu telah dikaji untuk mengesan gen toksin Shiga 1 dan 2 (stx1 dan stx2) dengan menggunakan multipleks rantaian tindak balas polimerase (PCR) dan pencirian dengan menggunakan cap jari Amplifikasi Polimorfik Asid Deoksiribonukleik-tindak balas rantaian polimerase (RAPD-PCR). Kesemua isolat telah diperolehi dari Makmal Sains Makanan dan Bioteknologi, Universiti Putra Malaysia Serdang, Selangor. Dalam pengesanan untuk gen stx1 dan stx2, 14 isolat (14/20) positif terhadap stx1 dan stx2. Manakala, 5 isolat (5/20) positif terhadap stx1 dan 1 isolat (1/20) negatif terhadap stx1 atau stx2 gen. Menggunakan analisis RAPD-PCR, 2 oligonukleotida telah dipilih kerana menghasilkan jalur yang jelas dan keputusan berulang. Mereka adalah OPAR8 (5'-TGGGGCTGTC-3') dan OPAR20 (5'-ACGGCAAGGA-3'). Oleh itu, kesemua 20 isolat E.coli O157:H7 telah disubtaipkan dengan menggunakan OPAR8 dan OPAR20. Primer OPAR8 menghasilkan 8 cap jari RAPD-PCR dinamakan P1 sehingga P11. Manakala OPAR20 menghasilkan 16 cap jari RAPD-PCR dinamakan Q1 hingga Q18. Gabungan dua primer dilakukan dengan menggunakan Kaedah Kumpulan Pasangan Tanpa penimbang dengan Keertian Aritmetik (UPGMA). Paparan dendrogram daripada analisis kelompok menunjukkan kesemua isolat E.coli O157:H7 boleh dibezakan kepada 20 isolat individu yang mencadangkan paras tinggi variasi genetik di geografi tempatan.

 

Kata kunci: Daging lembu runcit; Escherichia coli O157:H7; Gen Stx1 dan Stx2; PCR multipleks

 

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*Corresponding author; email: sahilah@ukm.my