MOLECULAR IDENTIFICATION OF A LOCAL LACTIC ACID BACTERIA (LAB) ISOLATE (B21) AND PRIMER CONFIRMATION FOR D-Lactate Dehydrogenase (D-LDH) GENE ISOLATION

Abstract

This study aims to identify a lactic acid bacteria (BAL) isolated  from native chicken (Gallus gallus domesticus) intestinum (B21) based on 16S rRNA gene sequence and also to confirm primers for PCR-based D-Lactate dehydrogenase (D-LDH) gene isolation. The genomic DNA of B21 was isolated then amplified using 16S rRNA. The PCR product then sequenced and aligned. The sequencing was done in 1^st Base Pte. Malaysia. The sequence then aligned using  the Basic Local Alignment Search Tool (BLAST) in NCBI to know the similar genome species. Isolation D-LDH gene was done through PCR using 5 primers (idb 0101, idb 1147, idb 0813, idb 1010, and idb 2021) of B21 and also J15 isolate genome. The PCR product were then sequenced and aligned with the D-LDH gene sequences in NCBI.

The results of molecular identification based on 16S rRNA markers showed that B21 has 99.45% similarity to Bacillus proteolyticus. Amplification of the LDH gene with idb 1010 primer on J15 isolate yielded 4 fragments (i.e. 650, 1400, 1500, and 1700bp). The results of phylogenetic tests showed that the 1700bp fragment has a high similarity to the D-LDH gene in Myobacterium tuberculosis  species with a bootstrap value of 84%. While other size fragments cannot be trusted to have similarity to LDH sequence because they have not reached  the minimum bootstrap  value that meets the qualifications.