optimized rtpcr assay for the detection of infectious myonecrosis virus (imnv)

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES Department of Aquatic Pathology OPTIMIZED RT-PCR ASSAY FOR THE DETECTION OF INFECTIOUS MYONECROSIS VIRUS (IMNV) By NGUYEN KHANH LINH A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Can Tho, December 2013 CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES OPTIMIZED RT-PCR ASSAY FOR THE DETECTION OF INFECTIOUS MYONECROSIS VIRUS (IMNV) By NGUYEN KHANH LINH A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture Science Supervisor Dr. TRAN THI TUYET HOA Can Tho, December 2013 ACKNOWLEDGEMENT First of all, I would like to express special thanks to my supervisor, Dr. Tran Thi Tuyet Hoa for her invaluable guidance, advice, and encouragement. I would also like to dedicate my great appreciation to Ms. Tran Thi My Duyen and Ms. Tran Viet Tien for her kind help in finishing the research. Many thanks are also given to all other doctors of the college of aquaculture and fisheries, and especially to those of the department of aquatic biology and pathology for providing me with great working and learning conditions. I would love to express my sincere appreciation to many of my friends, especially Duong Thanh Long, Hong Mong Huyen, Tran Thanh Can and Le Tan Hieu for their unconditionally kind help throughout the experimental period. Last but not least, I really want to thank my academic adviser, Dr. Duong Thuy Yen, who was guiding and encouraging me over the last four years, and my family for their great lifetime support which makes everything possible for me. The author, Nguyen Khanh Linh i APPROVE BY SUPPERVISOR The thesis “Optimized RT-PCR assay for detection of Infectious myonecrosis virus” which edited and passed by the committee, was defended by Nguyen Khanh Linh in 27/12/2013 Student sign Supervisor sign Nguyen Khanh Linh Tran Thi Tuyet Hoa ii ABSTRACT The purpose of this study is aim to apply RT-PCR procedure (OIE, 2009) for detection of Infectious myonecrosis virus on Penaeus vannamei. The procedure was amplified positive band at 328 bp in the first step and 139bp in the nested-step. The research was focused to optimize the nested-step with components reaction as 1X PCR buffer; 1.0mM MgCl2; 200µM dNTP mix; 0.465µM primer NF and 0.465µM primer NR; Taq polymerase 2.0U; 0.5µl template (cDNA). Thermal cycling PCR condition consisted of initial denaturation at 95ºC for minutes followed by 35 cycles of 95ºC for 30 seconds, 65ºC for 20 seconds, 72ºC for 30 seconds, 72ºC for minutes. Total amplification time is hours 11 minutes. Protocol also showed that the optimal RNA concentration using in study has given at 200ng/µl and 100ng/µl by sensitivity test. The specific test was also conducted with common virus in Penaeid shrimp as Monodon baculovirus, White spot syndrome virus, Infectious hypodermal and hematopoietic necrosis virus, Gill-associated virus. Besides, shrimp samples showed white necrotic muscles in the distal abdominal segments were also employed for the application test. iii Table of Contents ACKNOWLEDGEMENT I CHAPTER I INTRODUCTION 1.1 General introduction . 1.2 Research objective 1.3 Research activities CHAPTER . LITERATURE REVIEW 2.1 Overview global white leg shrimp industry . 2.2 Overview of white leg shrimp farming in Vietnam . 2.3 Common diseases in P.vannamei 2.3.1 Taura syndrome . 2.3.2 White spot disease . 2.3.3 Yellow Head Disease 2.3.4 Infectious hypodermal and haematopoietic necrosis disease 2.3.5 Infectious myonecrosis disease . 2.3.6. Diagnostic methods of IMNV 2.3.7 Research of IMNV detection by RT-PCR method . 10 2.4 Reverse transcriptase-polymerase chain reaction (RT-PCR) . 10 2.5 Factors affecting RT-PCR protocol . 13 2.5.1 RT primer 13 2.5.2 One-step and Two-step RT-PCR . 13 2.5.3 Quantitating the RNA 13 2.5.4. Check the RNA Integrity 13 2.5.5 Sequence of primer 14 2.5.6 Primer annealing . 14 2.5.7 Magnesium Concentration 14 2.5.8 Deoxynucleotide triphosphate (dNTPs) 14 2.5.9 Enzyme Taq pol 14 2.5.10 Thermal cycle 15 2.5.11 Primer concentration and DNA concentration 15 iv CHAPTER . 16 RESEARCH METHODOLOGY . 16 3.1 Research place 16 3.2 Materials . 16 3.3 IMNV infected shrimp: . 16 3.4 RNA extraction procedure: 16 3.5 Determining yield of RNA 17 3.6 cDNA synthesis with Randome Hexamers primer 17 3.7 Amplified components (IMNV detection followed by OIE, 2009) . 17 3.8 Electrophoresis 19 3.9 Test result 19 3.10 Determination of detection limit of the RT-PCR 19 3.11 Determination of specificity of the RT-PCR . 19 3.12 Amplification of IMNV from clinical samples by the RT-PCR (OIE, 2009) 20 CHAPTER . 21 RESULTS AND DISCUSSION 21 4.1 IMNV detection using OIE procedure (OIE, 2009) 21 4.2 Optimization of the chemical components and thermal cycles for the RT-PCR (IMNV - OIE, 2009) . 21 4.2.1 Optimization of the chemical components and thermal cycles of first step (IMNV - OIE, 2009) . 21 4.2.2 Optimization of the chemical components and thermal cycles of Nested step for the RT-PCR (IMNV - OIE, 2009) 22 4.2.2.1 Optimization of the chemical components and thermal cycles of Nested step for the RT-PCR (IMNV - OIE, 2009) . 22 4.2.1.2 Optimization of Taq polymerase concentration . 23 4.2.1.3 Optimization of the magnesium concentration and annealing time . 24 4.2.1.4 Optimization of the MgCl2 concentration 26 4.2.1.5 Optimization of the thermal cycles of nested step 28 4.3 Determination of detection limit of the RT-PCR 30 v 4.4 Determination of specificity of the RT-PCR . 31 4.5 Amplification of IMNV from clinical samples by the RT-PCR (OIE, 2009) . 32 CHAPTER . 33 CONCLUSIONS AND RECOMMENDATION . 33 5.1 CONCLUSIONS 33 5.2 RECOMMENDATION . 33 REFERENCES . 34 vi List of tables Table 3.1: Mixture A component . 17 Table 3.2: Mixture B component . 17 Table 3.3: First step RT-PCR component 18 Table 3.4: Nested RT-PCR component . 18 Table 4.1: PCR reagents of nested PCR for the detection of IMNV . 22 Table 4.2: PCR reagents of nested PCR for the detection of IMNV (change in Taq polymerase concentration) . 23 Table 4.3: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration and annealing time) . 24 Table 4.4: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration) . 26 Table 4.5: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration) . 26 Table 4.6: Optimization of RT-PCR assay for the detection of IMNV (number of cycles and final extension time) . 28 vii List of figures Figure 2.1 World shrimp aquaculture by species from 1991 to 2013 Figure 2.2 Shrimp aquaculture in Asia by species: 1991 - 2013 . Figure 2.3: The template RNA prior to initiating reverse transcription. . 10 Figure 2.4: Priming for reverse transcription . 11 Figure 2.5: First strand synthesis . 11 Figure 2.6: Removal of RNA . 12 Figure 2.7: PCR reaction 12 Figure 4.1: Result of 1st step of RT-PCR assay for the detection of IMNV 21 Figure 4.2: Result of 2st step of RT-PCR assay for the detection of IMNV 22 Figure 4.3: Result of 2st step of RT-PCR assay for the detection of IMNV (change in Taq polymerase concentration) 23 Figure 4.4: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration and annealing time) . 25 Figure 4.5: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration) . 27 Figure 4.5a: Magnesium concentration at 1.5mM . 27 Figure 4.5b: Magnesium concentration at 1.0mM . 27 Figure 4.6: Optimization of RT-PCR assay for the detection of IMNV (number of cycles and final extension time) . 29 Figure 4.7: Detection limit of the RT-PCR with dilution series of extracted RNA in agarose gel 30 Figure 4.8: Result of RT-PCR specific test with GAV, TSV, IHHNV, MBV infected samples . 31 Figure 4.9: PCR amplification of IMNV extracted from Penaeus vannamei . 32 viii CHAPTER RESULTS AND DISCUSSION 4.1 IMNV detection using OIE procedure (OIE, 2009) The IMNV infected sample was used to extract RNA and optimize for the RTPCR procedure (OIE, 2009) of IMNV detection. RNA extraction and determination of RNA concentration were conducted in this study, followed by the described protocols in 3.4 and 3.5 (Chapter 3). cDNA synthesis with Randome Hexamers primer was conducted in this study, followed by the described procedure in 3.6 (Chapter 3). cDNA temple was boiled in minutes prior to the addition to the first RT-PCR step that increased the sensitivity of detection (Polous and Lightner, 2006). 4.2 Optimization of the chemical components and thermal cycles for the RT-PCR (IMNV - OIE, 2009) 4.2.1 Optimization of the chemical components and thermal cycles of first step (IMNV - OIE, 2009) 328bp 200bp 100bp Figure 4.1: Result of 1st step of RT-PCR assay for the detection of IMNV Lane 1: 100bp DNA ladder (Promega) Lane 2: The product from the first step The result showed bright band at 328bp on agarose gel from the first step of IMNV detection procedure (RT-PCR: OIE, 2009) (Figure 4.1). From Figure 4.1, there was bright product at the expected size of 328bp. Therefore, the nested step was continues amplified from the first PCR product. 21 4.2.2 Optimization of the chemical components and thermal cycles of Nested step for the RT-PCR (IMNV - OIE, 2009) 4.2.2.1 Optimization of the chemical components and thermal cycles of Nested step for the RT-PCR (IMNV - OIE, 2009) Template for the nested reaction is the product from the first step reaction. The change of reaction component was displayed in table 4.1 Table 4.1: PCR reagents of nested PCR for the detection of IMNV Reagent Water x PCR buffer MgCl2 (25mM) dNTP mix (10mM each) Primer NF (100ng µl-1) Primer NR (100ng µl-1) Taq polymerase (5U µl-1) Template 1(cDNA) Concentration 1x 2.5mM 200µM total 0.465µM 0.465µM 2.5 U 100pg 200bp 139bp 100bp Figure 4.2: Result of 2st step of RT-PCR assay for the detection of IMNV Lane 1: 100bp DNA ladder (Promega) Lane 2: The product from the second step On an agarose gel, an optimized PCR amplification product gave bright band of expected size at 139bp (lane 2). However, there was an appearance of nonspecific product as a smear above the expected band. Magnesium concentration and polymerases were critical factors that affected successful of PCR. (Innis and Gelfand, 1990); (McPherson and Miller, 2006). 22 Different concentrations of Taq DNA polymerase may be required with respect to individual target template sequences or primers. Increasing the amount of Taq DNA polymerase beyond the 2.5 units/reaction in some cases increase PCR efficiency. However, adding more Taq DNA polymerase can increase the yield of nonspecific PCR products at the expense of the desired product. (Bartlett and Stirling, 2003). 4.2.1.2 Optimization of Taq polymerase concentration The change of reaction component was displayed in table 4.2 Table 4.2: PCR reagents of nested PCR for the detection of IMNV (change in Taq polymerase concentration) Reagent Concentration Water x PCR buffer 1x MgCl2 (25mM) 2.5mM dNTP mix (10mM each) 200µM Primer F (100ng µl-1) 0.465µM -1 Primer R (100ng µl ) 0.465µM Taq polymerase (5U µl-1) 2.0 U Template 1(cDNA) 100pg 200bp 139bp 100bp Figure 4.3: Result of 2st step of RT-PCR assay for the detection of IMNV (change in Taq polymerase concentration) Lane 1: 100bp DNA ladder (Promega) Lane 2: The product from the second step There was a bright band at 139bp and the smeared band was opaque. The result gave not significant brightness compared to the previous result (Figure 4.2). Taq polymerases is one of the most expensive and important chemical when optimizing PCR protocol (Coen, 2009). Taq DNA polymerase is the most commonly commercial enzyme used for PCR and it was used in this study. It is suitable for most 23 amplification reactions and already contained magnesium ion. Magnesium concentration is a critical factor affecting the success of PCR amplification because it may affect DNA polymerase activity and fidelity, DNA strand denaturation temperatures of both template and PCR product, primer annealing, PCR specificity, and primer-dimer formation (Bartlett and Stirling, 2003). Optimization of PCR thermal cycling conditions includes the determination of the cycle number, the temperature and incubation time period for template denaturation, primer annealing, and primer extension should be recommended after other chemical reactions were optimized (Bartlett and Stirling, 2003). Annealing time may affected optimizing, the longer annealing time, and the higher nonspecific products (McPherson and Miller, 2006). Taq concentration (2.0U) was kept but amounts of magnesium were reduced. 4.2.1.3 Optimization of the magnesium concentration and annealing time The change of reaction component and thermal cycles is displayed in table 4.3 Table 4.3: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration and annealing time) Reagent Concentration Water x PCR buffer 1x MgCl2 (25mM) 2.0mM dNTP mix (10mM each) 200µM -1 Primer F (100ng µl ) 0.465µM Primer R (100ng µl-1) 0.465µM -1 Taq polymerase (5U µl ) 2.0 U Template 1(cDNA) 100pg Perform an initial denaturation step: 95ºC for minutes Perform 39 cycles of PCR: 95ºC for 30 seconds 65ºC for 20 seconds 72ºC for 30 seconds 72ºC for minutes 24 200bp 139bp 100bp Figure 4.4: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration and annealing time) Lane 1: 100bp DNA ladder (Promega) Lane 2: The product from the second step The result showed the bright band at 139bp positive with IMNV. Reducing annealing time from 30 seconds to 20 seconds which showed significant different results. The yield of product was still high and nonspecific products were reduced when decreasing concentration of magnesium from 2.5mM to 2.0mM. Appearance of smear was reduced significantly in this experiment. According to A. Innis and H. Gelfand (1990), the magnesium concentration may affect primer annealing, strand dissociation temperatures of both templates and PCR products, product specificity, formation of primer-dimer artifacts, and enzyme activity and fidelity. Excess magnesium resulted in accumulation of nonspecific amplification products seen as smeared bands on an agarose gel, whereas insufficient magnesium results in reduced yield of the expected PCR product (Grunenwald and Haiying, 2003). The annealing time helped to form the ternary complexes at the correct template site, but too long annealing time created the opportunity for ternary complexes to form at incorrect binding sites (Subramanian, 2008). Thus, PCR protocols must adopt shortened annealing times to optimize the efficiency of annealing. This development showed the smear was darker than previous optimization though too much excessive product was generated. Chemical components needed to optimize continuously next time, especially magnesium concentration. The enzyme concentration is too high, so nonspecific background products may accumulate, and magnesium concentration may affect polymerases activation. The magnesium concentration would suggest reducing the yield of amplification products. 25 4.2.1.4 Optimization of the MgCl2 concentration The change of reaction component was displayed in table 4.4, 4.5 Table 4.4: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration) Reagent Concentration Water x PCR buffer 1x MgCl2 (25mM) 1.5mM dNTP mix (10mM each) 200µM Primer F (100ng µl-1) 0.465µM Primer R (100ng µl-1) 0.465µM -1 Taq polymerase (5U µl ) 2.0 U Template 1(cDNA) 100pg Table 4.5: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration) Reagent Concentration Water x PCR buffer 1x MgCl2 (25mM) 1.0mM dNTP mix (10mM each) 200µM Primer F (100ng µl-1) 0.465µM Primer R (100ng µl-1) 0.465µM -1 Taq polymerase (5U µl ) 2.0 U Template 1(cDNA) 100pg Perform an initial denaturation step: 95ºC for minutes Perform 39 cycles of PCR: 95ºC for 30 seconds 65ºC for 20 seconds 72ºC for 30 seconds 72ºC for minutes 26 Figure 4.5: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration) Figure 4.5a: Magnesium concentration at 1.5mM Lane 1: 100bp DNA ladder (Promega) Lane 2: The product from the second step 200bp 139bp 100bp a Figure 4.5b: Magnesium concentration at 1.0mM Lane 1: 100bp DNA ladder (Promega) Lane 2: The product from the second step 200bp 139bp 100bp b The RT-PCR optimizing results showed the positive band with IMNV at 139bp . It was noticed that both the smear and the yield were reduced slightly when reducing magnesium concentration but the expected band was also bright. In this experiment, only magnesium concentration was changed while other components and thermal cycles were the same. It is proved that magnesium concentration can cause accumulation of nonspecific products which were observed as smear on stain agarose gel. Decreasing amount of magnesium limited the yield of expected and nonspecific products. 27 Excessive magnesium can lead to the generation of nonspecific products in developing PCR. Magnesium ions interact with the DNA polymerase enzyme during the addition of enzyme base onto the end of an annealed primer. Because the close relationship between magnesium and Taq polymerases was said to be a cofactor of the polymerase enzyme, PCR amplification of a DNA target would not occur if magnesium were left out of the reaction (H. Stephenson and C. Abilock, 2012). Besides, there was a presence of magnesium ion in some commercial enzyme buffer so this concentration needs to be optimized to minimize error rates during the protocol (Wantprogress.com). The most frequent causes of PCR products smearing on a gel are excess templates or too many cycles (Innis and H. Gelfand, 1990). Extension time will play an important role in adjusting the outcome of the PCR reaction and artifacts are generally seen as smeared bands in agarose gels. The common final extension time was usually between minutes to 15 minutes because PCR needed more time for the polymerase molecules to complete synthesis of all the products (Grunenwald and Haiying, 2003). Nonspecific products that were generated may be the cause of smear existence. The number of cycles was also a critical parameter affecting yield and nonspecific amplification products. A common mistake is to execute too many cycles (Innis and H. Gelfand, 1990). 4.2.1.5 Optimization of the thermal cycles of nested step The change of thermal cycles was displayed in table 4.6 Table 4.6: Optimization of RT-PCR assay for the detection of IMNV (number of cycles and final extension time) Reagent Concentration Water x PCR buffer 1x MgCl2 (25mM) 1.0mM dNTP mix (10mM each) 200µM -1 Primer NF (100ng µl ) 0.465µM -1 Primer NR (100ng µl ) 0.465µM Taq polymerase (5U µl-1) 2.0 U Template 1(cDNA) 100pg Perform an initial denaturation step: 95ºC for minutes Perform 35 cycles of PCR: 95ºC for 30 seconds 65ºC for 20 seconds 72ºC for 60 seconds 72ºC for minutes 28 200bp 139bp 100bp Figure 4.6: Optimization of RT-PCR assay for the detection of IMNV (number of cycles and final extension time) Lane 1: DNA ladder (Promega) Lane 2: The product from the second step The expected band was observed at 139bp band, positive with IMNV. The smear was reduced significantly when the number of cycles decreased but final extension time increased. Not too much product was generated. The number of cycles was reduced from 39 cycles to 35 cycles, which showed that too many cycles can increase the amount and complexity of nonspecific background products. Too few cycles gave low product yield (Innis and H. Gelfand, 1990). Too many cycles was one of the reasons that formed nonspecific products as smear on agarose gel (Grunenwald and Haiying, 2003). Besides, increasing final extension time in order to enzyme had more time to synthesis completely all product (Grunenwald and Haiying, 2003). It was proven that too many cycles in the previous experiment produced too much excess and nonspecific products. The change of number cycles and final extension time had given specificity and fidelity band. 29 4.3 Determination of detection limit of the RT-PCR To determine suitable concentration of RNA used in study, RNA template was diluted into different concentrations including 200ng/µl, 100ng/µl, 10ng/µl, 1ng/µl, 100pg/µl and 10pg/µl 200bp 139bp 100bp Figure 4.7: Detection limit of the RT-PCR with dilution series of extracted RNA in agarose gel. Lane 1: 100bp DNA ladder Lane 2: IMNV control (-) Lane 3: sample with 200ng/µl of RNA extraction Lane 4: sample with 100ng/µl of RNA extraction Lane 5: sample with 10ng/µl of RNA extraction Lane 6: sample with 1ng/µl of RNA extraction Lane 7: sample with 100pg/µl of RNA extraction Lane 8: sample with 10pg/µl of RNA extraction The result showed the 139 bp band is positive with IMNV which proved the presence of IMNV in the sample (Figure 4.7). Two out of five samples were given bright bands at the expected size and it showed that concentrations at 200ng/µl and 100 ng/µl of RNA extraction gave expected results. It was noticed that OIE (2009) protocol can determine positive samples with IMNV at 200ng/µl and 100ng/µl while at lower concentration this determination could not be observed on agarose stained gel. According to Nguyen Van Thanh (2007), DNA template concentration affected significantly the amplification of PCR. 30 4.4 Determination of specificity of the RT-PCR The RT-PCR was amplified with common shrimp viruses as WSSV, MBV, IHHNV and GAV in order to determine the specificity of the test. 200bp 139bp 100bp Figure 4.8: Result of RT-PCR specific test with GAV, TSV, IHHNV, MBV infected samples Lane 1: IMNV control (+) Lane 2: IMNV control (-) Lane 3: 100bp DNA ladder Lane 4: Infected MBV sample Lane 5: Infected IHHNV sample Lane 6: Infected TSV sample Lane 7: Infected GAV sample The result showed that only samples containing infected shrimp with IMNV (+) gave bright band (lane 1) while infected sample with MBV (lane 4), IHHNV (lane 5), TSV (lane 6), GAV (lane 7) were gave negative results. It was proven that primer pair used in IMNV detection protocol (OIE, 2009) was only specific for IMNV. 31 4.5 Amplification of IMNV from clinical samples by the RT-PCR (OIE, 2009) To determine the application ability of the RT-PCR, five shrimp samples collected from Ca Mau and Bac Lieu provinces in Mekong Delta which had clinical signs of IMN disease were employed for the application test. 200bp 139bp 100bp Figure 4.9: PCR amplification of IMNV extracted from Penaeus vannamei Lane 1: 100bp DNA ladder Lane 2: IMNV control (-) Lane 3: IMNV control (+) Lane to lane 5: P. vannamei collected from Ca Mau Lane to lane 8: P. vannamei collected from Bac Lieu The result showed that all samples gave correct bands at expected size of 139bp. It was noticed that the reaction used correct and specific primers when amplifying DNA fragments of IMNV. Therefore, this protocol can be applied for the detection of IMNV in shrimp samples. 32 CHAPTER CONCLUSIONS AND RECOMMENDATION 5.1 CONCLUSIONS The research was focused to optimize the nested-step with components reaction as 1X PCR buffer; 1.0mM MgCl2; 200µM dNTP mix; 0.465µM primer NF and 0.465µM primer NR; Taq polymerase 2.0U; 0.5µl template (cDNA). Thermal cycling PCR condition consisted of initial denaturation at 95ºC for minutes followed by 35 cycles of 95ºC for 30 seconds, 65ºC for 20 seconds, 72ºC for 30 seconds, 72ºC for minutes. The RT-PCR procedure has showed the specificity to IMNV and has the sensitivity of 100ng/µl. 5.2 RECOMMENDATION To determine the stability of the procedure, the RT-PCR should be used to test more shrimp samples from different ponds, different stages from post larvae to adult shrimp. 33 REFERENCES Andrade T.P.D., Srisuvan T., Tang K.F.J. and Lightner D.V. (2007). 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(Eds.), Swimming through troubled water, Proceedings of the special session on shrimp farming, Aquaculture '95. 1-4 February 19995, o San Diego. World Aquaculture Society, Baton Rouge, LA, USA, pp. 84-94. Theresa Phillips, Requisites for Superior PCR Results, http://biotech.about.com/od/application/tp/SuccessfulPCR.htm 36 [...]... prior to the addition to the first RT-PCR step that increased the sensitivity of detection (Polous and Lightner, 2006) 4.2 Optimization of the chemical components and thermal cycles for the RT-PCR (IMNV - OIE, 2009) 4.2.1 Optimization of the chemical components and thermal cycles of first step (IMNV - OIE, 2009) 1 2 328bp 200bp 100bp Figure 4.1: Result of 1st step of RT-PCR assay for the detection of IMNV... The product from the first step The result showed bright band at 328bp on agarose gel from the first step of IMNV detection procedure (RT-PCR: OIE, 2009) (Figure 4.1) From Figure 4.1, there was bright product at the expected size of 328bp Therefore, the nested step was continues amplified from the first PCR product 21 4.2.2 Optimization of the chemical components and thermal cycles of Nested step for. .. thermal cycles of Nested step for the RT-PCR (IMNV - OIE, 2009) 4.2.2.1 Optimization of the chemical components and thermal cycles of Nested step for the RT-PCR (IMNV - OIE, 2009) Template for the nested reaction is the product from the first step reaction The change of reaction component was displayed in table 4.1 Table 4.1: PCR reagents of nested PCR for the detection of IMNV Reagent Water 5 x PCR buffer... 100pg Perform an initial denaturation step: 95ºC for 2 minutes Perform 39 cycles of PCR: 95ºC for 30 seconds 65ºC for 20 seconds 72ºC for 30 seconds 72ºC for 2 minutes 24 1 2 200bp 139bp 100bp Figure 4.4: Optimization of RT-PCR assay for the detection of IMNV (magnesium concentration and annealing time) Lane 1: 100bp DNA ladder (Promega) Lane 2: The product from the second step The result showed the bright... (Poulos et al., 2006) 1.2 Research objective The research aim to apply a specific and sensitive protocol for the detection of IMNV from Penaeus vannamei 1.3 Research activities This research was conducted two following contents:  Optimization of chemical ingredients of RT-PCR for the detection of IMNV  Optimization of cycling conditions of RT-PCR for the detection of IMNV 1 CHAPTER 2 LITERATURE REVIEW 2.1... chain reaction (PCR) is a technique for copying a piece of DNA a billion-fold As the name suggests, the protocol creates a chain of many pieces In this case the pieces are nucleotides, and the chain is a strand of DNA The purpose of this method is similar to the PCR except it allows amplification of small amounts of ribonucleic acid (RNA) RT-PCR is used for detecting viruses with an RNA genome and RNA... Centrifuge at 12000 x g for 10 minutes, remove the supernatant from the tube - Wash the pellet with 1ml of 75% ethanol per 1ml of TRIzol® Reagent used for homogenization - Vortex the sample briefly and centrifuge the tube at 7500x g for 5 minutes, then dry the RNA pellet Resuspend the RNA pellet in 40µl TE buffer, store at 80ºC 16 3.5 Determining yield of RNA - Use absorbance of RNA at 260nm to determine... 100bp Figure 4.3: Result of 2st step of RT-PCR assay for the detection of IMNV (change in Taq polymerase concentration) Lane 1: 100bp DNA ladder (Promega) Lane 2: The product from the second step There was a bright band at 139bp and the smeared band was opaque The result gave not significant brightness compared to the previous result (Figure 4.2) Taq polymerases is one of the most expensive and important... or reduced light microscopy may show loss of the normal striations Fragmentation of muscle fibres may also be apparent Squashes of the LO may show the presence of significant accumulations of spherical masses of cells (LOS) amongst normal LO tubules 9 The sensitivity was approximately tenfold lower than that of a one-step RTPCR assay using the same sample Other clinical methods such as clinical chemistry,... 100pg 1 2 200bp 139bp 100bp Figure 4.2: Result of 2st step of RT-PCR assay for the detection of IMNV Lane 1: 100bp DNA ladder (Promega) Lane 2: The product from the second step On an agarose gel, an optimized PCR amplification product gave bright band of expected size at 139bp (lane 2) However, there was an appearance of nonspecific product as a smear above the expected band Magnesium concentration and . farmed in super- intensive raceway systems in Hawaii (Brock et al., 1 983 ; Lightner, 1 983 , 1 988 ; Lightner et al., 1 983 a, 1 983 b; Brock and Lightner, 1990) in and widely distributed in cultured. Thuan and the Mekong Delta. In 2012, areas for P.vannamei farming increased 28, 169 ha (15. 5% total area), reaching 177 ,81 7 tons (3.2% production) compared to 2011, accounting for 5.9% cultural. 1996a; Lightner et al., 19 98; Lightner and Redman, 19 98) . Frozen imported commodity shrimp having the same diagnosis was reported in the United States (Nunan et al., 19 98; Durand et al., 2000).