20.109(S15):Diagnostic primer design (Day2)

Introduction

Today you will begin the second research project for Module 1. Specifically, you will design primers to test bird fecal samples for avian influenza virus (AIV) using a two-step real-time reverse transcription-polymerase chain reaction (RRT-PCR). AIV is found in several bird taxa; however, it is most consistently associated with waterfowl, gulls, and shorebirds. These birds are considered to be natural maintenance hosts of AIV, meaning that they have adapted to carry and transmit influenza strains. AIV can spread to domestic poultry and cause widespread outbreaks that result in serious disease. Furthermore, some AIVs have crossed the species barrier and caused disease in humans and other mammals. An example of an AIV crossing species barriers is the H5N1 subtype, which first infected humans in 1997 during a poultry outbreak. In fact, over the last century AIVs carried by wild birds have been implicated in the three major influenza pandemics. A better understanding of AIV abundance and epizootiology in wild birds will aid public health officials in the development of models that better predict human influenza pandemics.

To screen wild birds for AIV, embryonating chicken eggs can be used to incubate virus present in samples collected from birds. Following incubation, the viral genetic material is isolated and screened to identify AIV infected birds. Though this method is very sensitive, results may not be obtained for 1-2 weeks. To quicken the identification and screening process RRT-PCR was developed as a rapid and less expensive alternative. We will discuss this method in further detail on Day 5 of this Module. For today, your goal is to design the tools you will need for your RRT-PCR assay.

To examine the prevalence of AIV in wild birds in New England, you will screen genetic material isolated from fecal samples collected from a ‘scallop pile’ in Nantucket. In both projects for Module 1, you will use primers to amplify DNA. The primers you will use next time anneal to bacterial 16S sequences and will allow you to amplify this section of the genome for cloning. For this project, you will design primers that anneal to the matrix (MA) gene of the influenza A genome to enable the identification of AIV-infected birds.

Influenza A viral genomes are composed of eight segments of negative-sense, single-stranded RNA that encodes 11 proteins. Segments 1, 2, and 3 encode subunits of RNA polymerase. Discuss genes and proteins… Subtypes of influenza A are categorized according to the hemagglutinin (HA) and neuraminidase (NA) proteins, which are displayed on the surface of the viral envelope. There are 16 subtypes of HA and 9 subtypes of NA. In influenza A H5N1, the viral genome encodes HA subtype 5 and NA subtype 1. Because the MA gene is less variable, it is a better target to use for your assay.

Today you will begin the process of developing an assay that can be used to identify AIV. The first step is to design primers that are specific for the MA gene. Primers are short, single-stranded oligonucleotides that anneal to specific sequences of DNA or RNA. These small oligonucleotides can be generated to include any sequence and therefore can be designed to anneal to specific locations within an organisms genome. Due to this specificity, primers are very useful tools for developing assays that identify certain sequences of DNA or RNA.

Specifics for primer design…

Homework

1. Read the paper and guiding questions for our upcoming discussion on (Mod 1, Day 3). Spend just 10-15 minutes preparing a single slide for your part of the discussion. For now, aim for two things: (1) You should copy the figure you will be presenting into your slide, and (2) You should try to come up with a concise title that states the main conclusion to be drawn from that slide.

Protocols

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