Development of a PCR procedure for the detection of Baculovirus penaei in shrimp

An assay using a single-tube, 1-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) was established for the simultaneous detection of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV). Three primer sets, 9195 F/9992 R, 94 F2/R2, and ITS F/28S R, were mixed at a ratio of 3:1:1 to amplify specific fragments of the TSV, WSSV, and Penaeus vannamei genome, respectively, in the RT-PCR reaction. Shrimp samples were experimentally infected with WSSV and TSV. PCR-amplified products detected in the nucleic acid extraction of shrimp pleopods produced 4 kinds of results. With no virus infection, 1 fragment of 892 base pairs (bp) was amplified from a ribosomal RNA gene by primer set ITS F/28S R as an internal control. In samples only infected by WSSV or TSV, 2 fragments could be seen: either from WSSV (530 bp) plus the internal control or TSV (231 bp) plus the internal control, respectively. In cases of co-infection with both viruses, all 3 amplified products were detected simultaneously. This study is the first report of Penaeus vannamei specimens co-infected with WSSV and TSV being detected using a PCR method via experimental infection. KEY WORDS: RT-PCR · WSSV · TSV · Penaeid shrimp Resale or republication not permitted without written consent of the publisherDis Aquat Org 50: [9][10][11][12] 2002 wide host range among crustaceans (Flegel 1997) and distinctive clinical signs (white spots) in penaeid shrimps. The entire sequence of the double-stranded, circular DNA genome has been determined (van Hulten et al. 2001). TSV is a single-stranded RNA virus that causes serious disease in the PL, juvenile and adult stages of P. vannamei exclusively (Lightner & Redman 1998).Reverse transcription (RT) and polymerase chain reaction (PCR) technologies have proven to be powerful diagnostic tools for shrimp viral infections and for detection of viral reservoirs in asymptotic carriers. Methods for PCR diagnosis have been published for WSSV (Kimura et al. 1996, Lo et al. 1996, Takahashi et al. 1996, Kim et al. 1998, Tapay et al. 1999 and TSV (Nunan et al. 1998). This study was carried out to develop a modified method using a template comprised of total nucleic acid for simultaneous detection of WSSV and TSV in a single tube, 1-step multiplex RT-PCR. MATERIALS AND METHODS Experimental infections.Penaeus vannamei, weighing approximately 3.5 g and originating from a commercial shrimp hatchery in Tungkang, southern Taiwan, were reared from post larvae to the juvenile stage in an indoor recirculation system. Randomly selected specimens were checked using TSV RT-PCR (Nunan et al. 1998) and then WSSV PCR (Lo et al. 1996), and all were found to be PCR negative. Juveniles were stocked at a density of about 20 shrimp per 90 l aquarium tank. Inocula were prepared from patently infected (carapace with white spots) P. monodon for WSSV and from P. vannamei for TSV. Shrimp collected from cultivation ponds experiencing disease outbreaks in I-lan, north Taiwan, were proven to be virally infected by WSSV PCR ...

Section: Resultsmentioning

confidence: 99%

Simultaneous detection of white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) by multiplex reverse transcription-polymerase chain reaction (RT-PCR) in Pacific white shrimp Penaeus vannamei

Tsai¹,

Shiau²,

Lee³

et al. 2002

“…Although PCR is considered a very sensitive technique, it has several disadvantages. Crude homogenates of some shrimp tissues may contain interfering compounds that can lead to false positive or false negative results (Wang et al 1996, Nunan et al 2000. The appearance of early 2-step positive results in F5, F6, and F8 at 30, 45, and 30 DPS, respectively, followed by several negative test results may have been examples of 2-step PCR false positives.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of an immunodot test to manage white spot syndrome virus (WSSV) during cultivation of the giant tiger shrimp Penaeus monodon

Patil¹,

Palaksha²,

Anil³

et al. 2008

A monoclonal antibody-based immunodot test was compared to a polymerase chain reaction (PCR) assay for managing white spot syndrome virus (WSSV) on shrimp farms at Kundapur and Kumta situated in Udupi and Uttar Kannada Districts, respectively, of Karnataka on the west coast of India. Of 12 grow-out farms in Kundapur, 6 (F1 to F6) yielded shrimp samples that were negative for WSSV by both immunodot test and 1-step PCR from stocking to successful harvest. Samples from the other 6 farms (F7 to F12) were positive for WSSV by both immunodot test and 1-step PCR at various times post stocking, and their crops failed. In the 2 farms at Kumta (F13, F14), immunodot and 1-step PCR results were both negative, and harvests were successful. In contrast to 1-step PCR results, farms F5, F6, F13, and F14 gave positive results for WSSV by 2-step PCR, and they were successfully harvested at 105 d post stocking. Our results indicate that an inexpensive immunodot assay can be used to replace the more expensive 1-step PCR assay for disease monitoring.

“…It has become one of the most harmful viral pathogens to the worldwide shrimp industry (Rosenberry 2002). Diagnosis is based on clinical signs and laboratory tests, including histological analysis (Lightner 1996), Western blot and immunohistochemistry with monoclonal antibodies (Nadala et al 1997, Magbanua et al 2000, Nadala & Loh 2000, Poulos et al 2001, Anil et al 2002, dot-blot and in situ hybridization using gene probes (Durand et al 1996, Nunan & Lightner 1997, polymerase chain reaction (PCR) (Wang et al 1995, Lo et al 1996 and real time PCR (Durand & Lightner 2002). In most cases, the clinical signs such as lethargy, reduction in food consumption and reddish coloration are not pathognomonic (Lightner 1996).…”

Section: Introductionmentioning

confidence: 99%

Hyperthermia reduces viral load of white spot syndrome virus in Penaeus vannamei

Granja¹,

Vidal²,

Parra³

et al. 2006

We have previously reported that white spot syndrome virus-infected Penaeus vannamei (also called Litopenaeus vannamei ) maintained at 32°C show higher survival rates and a significant increase in number of apoptotic cells when compared to infected shrimp kept at 26°C. As apoptosis plays an important part in the antiviral response of invertebrates, we hypothesized that this process would reduce WSSV replication, allowing the shrimp to control the disease and survive. To test this hypothesis, shrimp were orally infected and maintained at either 26°C (Group 1) or 32°C (Group 2), DNA was extracted from haemolymph collected at various times from 6 to 216 h postinfection, and the number of viral units was quantified by real time PCR using SYBR Green. In parallel, histological examination was carried out to confirm the WSSV infection and to rule out concomitant diseases. Linear regression of real time PCR units (rtPCRU) of WSSV from Group 1 showed a significant increase with time post-infection (r 2 = 0.7383; p < 0.001). Conversely, there was no increase in rtPCRU with time post-infection in Group 2 (r 2 = 0.142), indicating that hyperthermia inhibited, either directly or indirectly, viral replication. In addition, comparison between the groups showed no difference in WSSV rtPCRU up to 48 h post-infection. After 72 h, shrimp from Group 1 had a significantly higher viral rtPCRU (ANOVA, p < 0.001). We conclude that hyperthermia-associated WSSV rtPCRU reduction could reflect either an increase in the shrimp antiviral response, or a direct negative effect on viral replication, or both. KEY WORDS: White spot syndrome virus · Real time PCR units · Viral load · Hyperthermia · Penaeus vannameiResale or republication not permitted without written consent of the publisher