Optimasi polymerase chain reaction (PCR) untuk deteksi Salmonella spp. pada udang segar

Abstract

Shrimp is an important protein source and non-oil commodity for foreign trade in Indonesia. Increase in the production volume for export and domestic demand is frequently challenged with rejection of shrimp exports to the importing country. This rejection is caused by many reasons, especially quality and sanitation, antibiotic residue and environmental issues. Presence of Salmonella spp. in shrimp has been frequently reported in Indonesia and worldwide. Within the last 5 years, USFDA recorded that 65% of the refusal of fishery products from Indonesia was due to Salmonella spp. The Indonesian National Standard 01-2728.1-2006 requires that Salmonella spp. on fresh shrimp should be negative in 25 g, because the bacteria are pathogenic and can cause health problems. Testing of Salmonella spp. in seafood is generally done using conventional methods that take 5 days. Various attempts were made to speed up the detection time of Salmonella spp., including by PCR. Variation of detection limits and accuracy for the detection of Salmonella spp. has been observed with different primers i.e. primers oriC targeting the replication gene of Salmonella spp., invA and invE (virulence genes), ompC which encodes outer membrane proteins and hns a DNA binding protein gene. This study aims to develop a PCR procedure for the detection of Salmonella spp. in shrimp, and compare it with conventional methods. The research was conducted in the following three phases: (a) optimization of PCR method with different annealing temperature (b) sensitivity and specificity assays of the PCR method and (c) detection of Salmonella spp. from shrimp using the PCR method selected and a conventional method. The results show that an annealing temperature of 640C is optimum to detect the 284 bp fragment of Salmonella invA gene. The PCR based detection method has a limit detection of 27.81 μg/mL and 1 cfu/mL of viable salmonellae with 100% specificity. The PCR based assay could detect 6 different Salmonella serovars (S. Enteritidis, S. Hadar, S. Heidelberg, S. Kentucky, S. Paratyphi and S. Typhimurium) and none of the non salmonellae isolates. Application of the PCR assay to detect Salmonella in shrimp after selective enrichment step suggested that all 16 samples are positive for Salmonella. Meanwhile the conventional method only detected 3 samples as positive for Salmonella. Use of a primer targeting invA gene is potential for detection of salmonellae in shrimp.