Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Multiplex-pcr Protocol Development for Rapid Screening of White Spot Syndrome Virus (wssv) in Shrimp

Sharmin Aktar
Department of Fisheries, Upazila Fisheries Office, Shalikha, Magura, Bangladesh

Md. Shahin Parvez
Fisheries and Marine Resource Technology Discipline, Khulna University, Khulna 9208, Bangladesh

H M Rakibul Islam
Bangladesh Fisheries Research Institute, Shrimp Research Station, Bagerhat, Bangladesh

Md. Nazmul Ahsan
Fisheries and Marine Resource Technology Discipline, Khulna University, Khulna 9208, Bangladesh

Abstract/ Executive Summary:

This study was aimed to develop a faster single step multiplex PCR protocol for the simultaneous detection of white spot syndrome virus (WSSV) with its host (i.e. shrimp) as internal positive control. To do so, four combinations of primer were tested (I. 16S rRNA+Lo F1R1; II. 16S rRNA+Lo F2R2; III. 16S rRNA+Lo F1R2; IV. 16S rRNA+Lo F2R1) which were selected based on two pairs of WSSV specific primer (Lo F1R1 and Lo F2R2) and one pair of shrimp specific primer (16S rRNA). DNA extracted from WSSV infected shrimp were amplified by PCR in a single tube using each of the primer combinations and the thermal cycling conditions as well as reagent compositions were optimized. All the primer combinations yielded their expected band sizes with stronger band resolution intensity that indicated the development of four multiplex PCR protocols. The developed multiplex protocols reduced the chance of cross-contamination and these were found to be faster, single step and unique with less effort and resource use. Considering sensitivity and specificity, among the protocols, we suggested the protocols based on 16S rRNA+Lo F1R1 and/or 16S rRNA+Lo F2R2 primer combinations for rapid and routine screening of WSSV in shrimp PL, juvenile and adult.

Keywords: Faster; Multiplex PCR; Shrimp; Simultaneous detection; Single step; WSSV

Location: Biochemistry and Molecular Genetics (BMG) laboratory of Fisheries and Marine Resource Technology (FMRT) Discipline of Khulna University, Bangladesh.

Start Date: 00-07-2014

End Date: 00-06-2015

Program Area: Animal Health and Management

Commodity: Shrimp, Virus

Objectives:

This study was aimed at optimizing a single-step multiplex PCR protocol that can be conveniently used for simultaneous and rapid screening of WSSV presence in shrimp thereby reducing the overall reaction time, reagent amount, and chance of carry-over contamination.

Methodology:

Study period and location- The study was conducted during July 2014 to June 2015 at Biochemistry and Molecular Genetics (BMG) laboratory of Fisheries and Marine Resource Technology (FMRT) Discipline of Khulna University, Bangladesh. Sample collection and processing- Fifty juvenile/adult tiger shrimp (Penaeus monodon) having apparently gross external symptoms of white spot disease were collected by preserving in 70% ethyl alcohol (Hossain et al. 2004) from the shrimp ponds located at Satkhira district, a coastal district of Bangladesh, devoted to shrimp viral diseases endlessly (Islam et al. 2007). About 25 – 30 mg tissue from the pleopods of collected shrimps were dissected separately with sterilized surgical blades to avoid cross-contamination and placed into a 1.5 ml sterile micro-centrifuge tube. Total genomic DNA extraction- Sodium dodecyl sulfate (SDS) based DNA extraction protocol previously developed by our laboratory was followed for the extraction of total genomic DNA from tissue samples. Briefly, samples were homogenized and lysed by 500 μl of 1% SDS based DNA extraction buffer (10 mM Tris-HCl (pH 8), 25 mM EDTA, 100 mM Nacl, 1% SDS) which followed centrifugation at 12000 rpm for supernatant collection (~350 μl). DNA of the supernatant was precipitated by adding 700 μl (two volumes of the supernatant) 100% ethanol and 75% ethanol was used to wash the precipitated DNA. Finally, the ethanol was removed by pipetting/decanting carefully. The DNA pellets into the tubes were air-dried immediately by keeping tubes open at room temperature for 10 – 15 min. The extracted DNA inside the tube was solubilized with 100 μl TE buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA) by slowly passing the pellet through a pipette tip. DNA quantity and quality was measured spectrophotometrically according to Strauss (1998) where DNA quantity was measured photometrically at 260 nm wavelength (OD260) and quality was determined by measuring the ratio of one more photometric value at 260 nm wavelength (OD260/ OD280). Primer Selection and combination- One pair of shrimp-specific (host / internal positive control) primer and two pairs of WSSV specific primer were taken into consideration. The WSSV specific primers correspond to a cloned, 1461 bp SalI-digested WSSV genome fragment as described by Lo et al. (1996) where Lo F1R1 and Lo F2R2 act as the outer primer and inner primer respectively. Using both of the WSSV specific primer, two more primer pairs were also theoretically obtained in a factorial design. To develop the multiplex protocol, four primer combinations were finally selected using the WSSV and shrimp-specific primer pairs as depicted. Testing of the major primers by PCR- Prior to the development of multiplex PCR protocol, the three major primer pairs were tested separately in separate tube employing single-step PCR. A 10 µl reaction mixtures for each specific tube was prepared with a combination of appropriately diluted approximately 10 – 25 ng sample tissue DNA as the templates with 2 µM of each primer (F / R) and 5 µl of premix (1X PCR buffer, 2 mM MgCl2, 200 µM dNTP’s and 0.25 unit Taq polymerase enzyme; TAKARA BIO INC., Japan) in 0.2 ml thin-walled PCR tubes. PCR thermal cycling conditions were empirically determined based on the previous studies (Lo et al. 1996; Islam et al. 2007). Thermal cycling conditions that were tested ranged 25 – 30 sec at 95°C for the first round followed by 30 cycles of denaturation for 30 – 40 sec at 94°C, 25 – 30 sec of annealing at 54 – 62°C and 40 – 45 sec of extension at 72°C. The last extension step at 72°C was extended for 5 min. Optimization of the multiplex PCR protocol- Both WSSV DNA and shrimp DNA (host/internal positive control) were amplified simultaneously in a single tube using each pair of the four (04) combinations of primer. All the PCR assays for the multiplex protocol were done with a 10 µl PCR cocktail in a single tube using a thermocycler (BioRed C1000, Singapur). The multiplex PCR protocol was developed by manipulating the primer and template concentrations, thermal cycling conditions (temperature and time) and reagents compositions found for the separate primer optimization. If all the conditions were found to be optimum for the protocol, only annealing temperatures were empirically altered to obtain the best banding pattern with stronger intensity. Appropriate control reactions (reagent control and negative control) were run in parallel to rule out the possibility erroneous amplification or no amplification at all. The PCR mixture being all the reagents without primers was considered as reagent control and the PCR mixture being all the reagents with DNA of tilapia species replacing shrimp DNA was considered as negative control. All the amplified PCR products were run by electrophoresis in TAE buffer on 1.5% agarose gel mixed with ethidium bromide. The banding pattern, intensity and sizes were identified in comparison with the 100 bp DNA ladder by visualizing under UV trans-illuminator.

Source of Information: Journal of Fisheries, Volume 8, Issue 3, December 2020, Pages 885–890

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The study optimized four multiplex PCR protocols target-ing both WSSV and shrimp DNA; each of which was optimized employing a single tube in a faster, non-stop single-step PCR with less resource and time use. However, considering sensitivity and specificity, the protocols using the primer combination of 16S rRNA+Lo F1R1 and/or 16S rRNA+Lo F2R2 are suggested to follow for rapid and routine screening of WSSV in shrimp

Knowledge Management: Journal