Optimization of Polymerase Chain Reaction (PCR) Primers for Early Detection of Quagga and Golden Mussels

Project ID: 3688
Principal Investigator: Jacque Keele
Research Topic: Invasive Species
Priority Area Assignments: 2014 (Zebra and Quagga Mussels)
Funded Fiscal Years: 2014
Keywords: invasive species, invasive mussels, golden mussels, polymerase chain reaction, dna detection

Research Question

The early detection of invasive mussels is important for maintaining Reclamation facilities and waters, as invasive mussels can destroy infrastructure as well as disturb the local biota and water chemistry of a reservoir. Currently, Reclamation primarily focuses on the intrusion of zebra and quagga mussels in the western United States, but Reclamation would benefit from also focusing on the early detection of golden mussels in the United States, as it will likely become a problematic invasive organism. The larval (veliger) life stage of these mussels can be identified using microscopy; however polymerase chain reaction (PCR) has proven to be an invaluable tool for monitoring raw water samples for invasive mussel DNA.
Reclamation currently utilizes PCR alongside microscopy to detect invasive mussel veliger DNA in raw water samples. Reclamation has worked to optimize the PCR process for early detection of invasive mussels, but the process could be further improved by the creation and implementation of secondary primers. The creation of more specific secondary primers for quagga and golden mussels will hopefully increase the sensitivity of the assay, providing a more accurate test.
Once the secondary primers have been created the efficacy of the primers will be compared to the original Cox1 primers. The primers will be tested on small numbers of veligers of each life stage. This test will help determine the amount of tissue required for the assay to produce a positive test result.
This research includes three goals: the first is to provide a comprehensive literature review of current DNA extraction, PCR techniques, and available primers for quagga and golden mussels, the second is to develop secondary PCR primers for quagga and golden mussels, and the final objective is to compare the efficacy of the quagga and golden mussel published and secondary primers by determining the amount of tissue needed for detection in samples with and without organic inhibitors.

Need and Benefit

It is important to improve the efficiency and accuracy of quagga and golden mussel detection methods so that the massive early detection effort is worthwhile. One of the ways that Reclamation is improving detection ability is through the implementation of DNA testing. Polymerase chain reaction (PCR) is a molecular method that can be used to test raw water samples to determine the presence of invasive mussel DNA. Reclamation currently has in place a PCR assay that detects the cytochrome oxidase 1 (COX1) gene from quagga and zebra mussel tissue, and primary primers have already been developed for the golden mussel. PCR is a dynamic process, and although Reclamation has made great strides in optimizing the PCR method for early detection, further research on the development of additional secondary primers should be done in order to increase the robustness of the test.
Primer effectiveness can be determined by testing their ability to pick up small amounts of target DNA. The amount of tissue required for a positive PCR result is currently unknown. A single veliger may only have one or two copies of the target gene, which may not be enough genetic material for amplification. This research will help determine the limits of the PCR primers at detecting low numbers of mussels, and mussels of variable size classes. This research will also help to determine how organic material in a sample inhibits primer function. Reclamation's early mussel detection program and water managers will benefit from this research because PCR results can be better correlated to the amount of veligers that were present in the sample.
Reclamation's early detection program for quagga and zebra mussel veligers is one of the leading programs in the west. Reclamation is also preparing for a possible invasion by the golden mussel, which may become a threat to western waterways. Researchers have shown that there is a high probability that the golden mussel will spread to North America. The golden mussel can tolerate higher salinity than the quagga mussel, and has lower calcium and pH requirements for survival. Many of the waterways in North America will be ideal habitat for golden mussel populations. Aggressive populations of golden mussels have already developed in Brazil, and Reclamation has been working to adapt a PCR method for the early detection of golden mussels with scientists from CETEC-Fendacão Centro Tecnológico de Minas Gerais, Brazil. Early detection of golden mussels in North America will allow Reclamation to track the spread and work with state and federal managers to control golden mussels, before they become problematic.

Contributing Partners

Contact the Principal Investigator for information about partners.

Research Products

Bureau of Reclamation Review

The following documents were reviewed by experts in fields relating to this project's study and findings. The results were determined to be achieved using valid means.

Optimization of Early Detection of Quagga Mussels by Polymerase Chain Reaction (final, PDF, 1.4MB)
By Jacque Keele
Report completed on December 11, 2014

The optimization of PCR for quagga mussel early detection is an ongoing process. Understanding the importance of a quality control/ quality assurance plan has been important to maintaining good laboratory practices. Many different aspects of the PCR process have been explored: determining the best sample storage conditions, optimizing the DNA extraction, and the PCR master mix, and primers used for amplification. Also, determining how humic acid affects the PCR reaction was assessed to determine

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Last Updated: 6/22/20