A COMPARATIVE STUDY ON COLONIC AND MILK BACTERIA BY DOUBLE-DIGESTED 16S RRNA GENE AMPLICONS

Abstract

16S rRNA gene was amplified from faecal or milk total genomic DNA of eight women by polymerase chain reactions (PCR) using a pair of bacteria specific universal oligonucleotide primers. Amplification products were digested with both HaeIII and TaqI restriction enzymes. Sampling period covered the second and third trimester of pregnancy and further 6 months after birth for the collection of breast milk. Digestion products were resolved in an 8% polyacrylamide gel and restriction fragment length polymorphism (RFLP) profiles were visualized by silver staining. A control group was also prepared from eight other women who were single and without children. In the RFLP profiles, clearly discernible 66 digestion products (bands) were selected and subjected to unweighted pair-group method with arithmetic mean analysis (UPGMA). Results indicated that almost 58% of the clustering comprised common banding patterns, shared by faecal, milk and control samples. Nine of the clusters (approximately 13%) were exclusive to faecal and milk samples. Prominent bands present in the milk RFLP patterns indicated that four of the mothers who adopted traditional food sources could have almost identical prominent bacteria in their milk.