DETEKSI DINI Enterocytozoon hepatopenaei (EHP) PADA UDANG VANAME (Litopenaeus vannamei) MENGGUNAKAN METODE PCR (POLYMERASE CHAIN REACTION)

Abstract

ABSTRAKSejak akhir tahun 2014, wabah kotoran putih atau yang sering disebut juga WFD (White Feces Disease), merupakan salah satu masalah yang sering terjadi pada petambak udang di Indonesia. Wabah ini diketahui disebabkan oleh Enterocytozoon hepatopenaei (EHP) dan telah mengakibatkan retardasi pertumbuhan hingga kematian pada udang. Hingga saat ini, penyakit WFD dapat dideteksi dengan cara uji histologi, hibridisasi in situ, dan PCR. Penelitian ini bertujuan untuk mendapatkan metode deteksi dini penyakit EHP pada udang vaname dengan metode PCR melalui perancangan primer yang spesifik dan sensitif. Pada penelitian ini dilakukan isolasi EHP pada udang vaname yang terinfeksi, kemudian dideteksi dengan metode PCR yang mentarget SWP (spore wall protein) dari EHP serta pengujian spesifitas dan sensitivitasnya. Hasil yang diperoleh menunjukkan bahwa EHP dapat diisolasi dari udang yang terinfeksi dan dapat didesain dua pasang primer yaitu SWP-EHP1 dan SWP-EHP3 yang mentarget spore wall protein EHP. Kedua primer ini dapat digunakan untuk deteksi EHP menggunakan PCR, dengan produk PCR pada primer SWP-EHP1 yaitu 398 bp dan primer SWP-EHP3 sebesar 415 bp, serta nilai suhu annealing optimal pada 48 o C.Hasil pengujian sensitivitas primer, diketahui bahwa primer SWP-EHP1 dapat mendeteksi EHP hingga jumlah DNA target sebanyak 7,74 x 10 2 kopi sedangkan primer SWP-EHP3 dapat mendeteksi hingga 16,2 x 10 2 kopi. KATA KUNCI: udang vaname; WFD; Enterocytozoon hepatopenaei; PCR ABSTRACT: Detection of EHP (Enterocytozoon hepatopanaei) from whiteleg shrimp (Litopenaeus vannamei) by Polymerase Chain Reaction (PCR) method. By: Annisa Fitriah Faisal and Adi PancoroSince 2014, white feces disease (WFD) is one of the emerging problems for whiteleg shrimp farming industries in Indonesia. This outbreak is known to be caused by Enterocytozoon hepatopenaei (EHP) infection to shrimp. EHP infection resulted in growth retardation to a mass mortality in shrimp. To date, WFD can be detected by histology, in situ hybridization and PCR. This study aimed to obtain an early detection method of EHP on whiteleg shrimp by PCR method through specific and sensitive primers design. In this study, we isolated the DNA of EHP from infected whiteleg shrimp, then detected by PCR method which targeted spore wall protein (SWP) from EHP as well as sensitivity and specificity testing. As a result, EHP can be isolated from infected shrimp and can be designed 2 pairs of primers (SWP-EHP1 and SWP-EHP3) targeting spore wall protein of EHP. These primers could be used for EHP detection using PCR, with PCR products from primers SWP-EHP1 was 398 bp and from SWP-EHP3 primers was 415 bp, with an optimum annealing temperature of 48 o C. Primers sensitivity test results revealed that primers SWP-EHP1 could detect EHP to 7.74 x 10 2 copies while the primers SWP-EHP3 could detect up to 16.2 x 10 2 copies.

Mini-review utilization of Vibrio parahaemolyticus virulence coding genes for early detection of acute hepatopancreatic necrosis disease (AHPND)

“…The validity of Vibrio parahaemolyticus AHPND (Vp AHPND) strain detection in whiteleg shrimp (Litopenaeus vannamei) in Indonesian shrimp culture was successfully performed employing the real-time PCR method. This step was carried out through several stages including the testing of specificity, sensitivity, repeatability, reproducibility, and sampling field tests in shrimp ponds (Nainggolan et al, 2020). The analytical performance test showed that the application of real-time PCR method using VpPirA primer was effective in detecting Vp AHPND strain and showed a sensitivity up to 10 copies µL.…”

“…Vibriosis has caused mass mortalities in all shrimp stages, from nauplius, zoea, mysis, and post-larvae to adults in rearing ponds. Vibriosis disease in seedling segmentation is often associated with luminous Vibrio harveyi or while in the pond rearing stage it is caused by non-luminous V. parahaemolyticus (Fuandila et al, 2019;Munaeni et al, 2020;Nainggolan et al, 2020). In previous studies, we have identified the important genes of the pathogens which is very crucial for developing the rapid and early diagnosis tools for the disease detection (Kadriah et al, 2011;Kadriah et al, 2014;Setiawan et al, 2015;Nainggolan et al, 2020).…”