DEVELOPMENT OF REAL TIME POLYMERASE CHAIN REACTION FOR DETECTION OF Salmonella typhimurium AND Salmonella enteritidis IN FISH

Abstract

Previously designed endpoint PCR has been adapted for use with real time PCR to detect the presence of Salmonella typhimurium and Salmonella enteritidis in fish. Optimization of a standard curve in the presence of herring sperm DNA as background matrix indicated that the real time PCR highly efficient with the Pearson coefficient of determination (R2) value = 0.99937 and slope (M) value = -3.44. An enrichment method (overnight culture) significantly increased (p<0.05) the sensitivity of real time PCR. Comparison of real time PCR and the conventional isolation method based on biochemical tests has been conducted. In terms of their sensitivity, real time PCR and the conventional methods are not significantly different in the level of confidence 95%. Both real time PCR with enrichment method and conventional biochemical method can detect the presence of Salmonella spp. in spiked sample. However the direct extraction method was only detecting the presence of Salmonella in higher concentration. While the sensitivity both conventional and real time PCR are similar, the real time PCR has an advantage to detect the pathogen qualitatively and quantitatively depending on processing method.

Pengembangan Prekultur Oxgall sebagai Sampel Klinis untuk Deteksi Salmonella typhi dengan Metode Real-time PCR

“…Kehadiran DNA dapat diamati pada grafik yang muncul sebagai hasil akumulasi fluoresensi probe (penanda). 12,13,14,15,16 Berdasarkan proses Real-time PCR, optimasi spike Real-time PCR penting dilakukan untuk mengetahui reagen yang digunakan baik untuk spesimen klinis. Namun, jumlah bakteri yang terlalu rendah dalam darah pasien tifoid (diperkirakan rata-rata 0,3 CFU / mL darah) membuat S. typhi sulit untuk dideteksi dengan Real-time PCR.…”

DESAIN TAQMAN PROBE SECARA IN SILICO SEBAGAI PENDETEKSI MUTASI PADA KODON 516 GEN rpoB Mycobacterium tuberculosis UNTUK METODE REAL-TIME PCR

“…Untuk mengatasi resistensi pada kasus MDR-TB, maka diperlukan suatu metode deteksi molekular yang cepat dan dapat mendeteksi mutasi secara spesifik, sehingga pasien akan memperoleh terapi yang tepat. Metode deteksi molekular yang digunakan yaitu real-time PCR, dengan keunggulannya dapat mendeteksi patogen secara kualitatif maupun kuantitatif (Ramirez et al, 2010;Siregar et al, 2012). Pada metode realtime PCR dibutuhkan DNA probe yang dapat mendeteksi mutasi secara spesifik dengan adanya label fluoresen, yaitu pada ujung 5' dan ujung 3'.…”

Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis

“…For PCR-based methods, the pathogen needs to be grown and a high concentration of nucleic acid is required to be extracted. 11 , 12 Bacterial flagellin is one of the outer membrane proteins that serve many functions like mobility, pathogenicity, and adjuvanticity and shows toll-like receptor (TLR)-ligand activity. It is effective at very low doses 13 , 14 and binds to toll-like-receptor 5 (TLR 5), which is present on the immune-system cells (epithelial cells, dendritic cells, and macrophage).…”