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September 18, 2013
Everything You Ever Wanted to Know about PCR
But Were Afraid to Ask
The September/October 2013 issue of The Global Aquaculture Advocatethe bimonthly magazine of the Global Aquaculture Alliance, contains a long, technical article about polymerase chain reaction (PCR) assays for the whitespot virus (WSSV). Scientists at Taiwan’s GeneReach Biotechnology Corp., which markets a machine for doing fast, accurate, pond-side tests for shrimp viruses (see advertisement below), wrote the article. Here are some excerpts:
Polymerase Chain Reaction
Polymerase chain reaction (PCR) assays are currently the most accepted molecular method for the detection of shrimp whitespot disease.
PCR primers identify the target specifically by recognizing novel signatures found within the genetic materials of target organism.
After target amplification, the reactions need to be processed manually for signal detection in some assays. In addition, these assays normally require hands-on time to assemble the reactions before the target amplification reaction. Because these methods involve highly sensitivity laboratory operations, protocols need to be implemented to minimize risks of cross contamination, which can lead to false positive or negative results.
Types of PCR Assays
Commercial WSSV assays are based on different PCR methods with minor variations in the reaction device and mechanisms of signal generation and detection. These methods include “one-step” PCR, nested PCR, real-time quantitative PCR (qPCR) and insulated isothermal PCR (iiPCR).
One-Step PCR Assays: Conventional one-step PCR assays include only basic amplification of target DNA, leading to sensitivity lower than that of the other PCR formats. Product detection relies mainly on gel analysis, which involves the separation of products through a gel of certain pore sizes in an electronic field (gel electrophoresis) and staining and visual detection of product bands on the gel with the help of a fluorescent dye.
Target amplification and product detection usually take two to three hours to complete. Besides being labor-intensive and time-consuming, these procedures can bring risks of cross contaminating amplicons into future assays, leading to false positive results. However, assays based on this format are generally cheaper than the other PCR assays.
Nested PCR Assays: To improve the sensitivity and specificity of one-step PCR, products of the first assay can be amplified further in a nested, second round, of PCR. This type of assay requires more hands-on time than the other methods because it includes two assembly steps and usually requires gel analysis of reaction products.
Target amplification and product detection (gel analysis) usually take three to four hours. However, the possibility of semi-quantitative analysis of WSSV provides important information about viral loads in shrimp samples to follow the progression of the disease. This is currently the method most recognized and accepted by the shrimp farming industry because of its satisfactory sensitivity, acceptable costs and reasonable sample throughput. Among the commercial WSSV kits, the IQ2000 WSSV Detection and Prevention System has been certified by the World Organization for Animal Health (OIE) as fit for the diagnosis of whitespot disease in crustaceans.
Real-Time Quantitative (qPCR) PCR Assays: In real-time qPCR, integration of a fluorescent dye/probe in the PCR reaction and optical detection module in the PCR apparatus enable easy qualitative and/or quantitative detection of amplicon production. However, real-time qPCR requires a sophisticated and expensive device for reaction and signal detection.
Inclusion of fluorescent dye-linked probes makes these assays more sensitive, yet more costly than single-step PCR. The advantages of real-time qPCR include the fact that results can be obtained without opening the reaction tubes, greatly reducing cross-contamination risks that come with gel analysis of PCR products. Furthermore, real-time qPCR usually takes one to two hours to complete and allows quantitative detection of WSSV to assist assessment of the severity of disease outbreaks. Capable of high-throughput analysis, real-time qPCR can fulfill the need of large-scale farms to screen large numbers of samples in a PCR lab.
Insulated Isothermal (iiPCR) PCR Assays: The iiPCR assay is based on a new PCR format that requires only a simple heat source to drive the PCR cycles. It works in a portable nucleic acid analyzer, designed specifically for iiPCR with capability to detect products automatically and make pond-side detection of WSSV possible. It is similar to real-time qPCR in providing automatically processed results of high sensitivity without any post-reaction steps.
Automatic reading of product signals makes the assay reliable and user-friendly, and reduces human errors and discrepancies in data interpretation. iiPCR assays can be done in approximately 1.5 hours. The costs of reagents and devices for iiPCR assays are lower than those for real-time qPCR assays. However, iiPCR assays do not provide quantitative scores, and the devices can process eight reactions a minute. The only iiPCR-based assay for WSSV, LCi Plus WSSV Kit with POCKIT System, was recently certified by the OIE as fit for the diagnosis of whitespot syndrome.
Sensitive and specific detection of WSSV at appropriate check points is critical for all shrimp farming facilities. Depending on the budget, location and resources of the facility, different PCR assays can be selected to fulfill the needs for WSSV detection.
For large-scale farms that hire a specialist and can afford real-time PCR reagents and machines, real-time qPCR assays are recommended because of their high-throughput capacity and short hands-on time. Nested PCR assays provide satisfying results for facilities with limited budgets. On the other hand, the portable and user-friendly iiPCR assays are suitable for small-scale farms, where only a small number of samples need to be tested at each checkpoint.
Information: Dr. Pei-Yu Alison Lee, GeneReach Biotechnology Corp., No. 19, Keyuan 2nd Road, Central Taiwan Science Park, Taichung, 407 Taiwan (phone 886-4-24639869, fax 886-4-24638255, email firstname.lastname@example.org://www.iq2000kit.com/news_detail.php?gid=2&nid=30
Sources: 1. The Global Aquaculture Advocate (The Global Magazine for Farmed Seafood). Editor, Darryl Jory (email@example.com). Innovation/PCR-Based Tools Aid Biosecurity Via Detection of White Spot Syndrome Virus. Dr. Pei-Yu Alison Lee, Dr. Ping-Hua Teng and Simon Chung. Volume 16, Issue 5, Page 104, September/October 2013. 2. The Global Aquaculture Advocate. Advertisement: GeneReach Biotechnology Corp. Volume 16, Issue 5, Page 105, September/October 2013.
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