You may discuss the questions with others or look up any resources. But, you must work on your answers in your own language. No plagiarism, please.
I don’t expect much details. Add details only when necessary. Diagram is always a good idea.
There is no fixed answers. You can give answers different from what I have in mind. However, I will pay attention to the scientific reasoning supporting your answers.
Q1. You have developed a quantitative real-time PCR assay for detection of Clostridium perfringens. Initial testing of the protocol showed that it works perfectly well when evaluated using isolated C. perfringens strains. However, when applied to the samples from chicken farms, the detection sensitivity was not good enough (e.g. many samples gave Ct value >37 cycles, giving false-negative results). How would you improve the detection sensitivity of your Quantitative RT-PCR assay? (Think about nested PCR!)
Q2. Our lab recently performed an experiment to study a potential microbiota present in chicken egg embryos. All culture results were negative (both aerobic and anaerobic), but PCR showed positive DNA amplification of 16S rRNA genes. What are the all possible ways to interpret the result? Please provide scientific basis supporting each point of your arguments.
Q3. You are given a project to develop a multiplex PCR assay for simultaneous detection of all 3 species (e.g. species A, B, and C) that belong to a previously uncharacterized genus Calmonella (there is no such thing. I made up!). You have previous experience in the following typing methods: PCR-RFLP, MLVA, Rep-PCR, AFLP, and MLST. You decided to use one of these typing methods, and use the resulting data as the basis for primer design for multiplex PCR. Which strain typing method would give the most informative result that would allow you to design PCR primers specific for each of the species A, B, and C? Pick one method, and provide your scientific reasoning. Explain how you would use the typing result to design primers. Use hypothetical result to illustrate your points.
Q4. You want to develop a rapid quantitative assay for specific detection of a newly discovered bacterial species, which has been shown to be extremely pathogenic to humans. You found that the bacterial cells of this species are extremely difficult to break open to release DNA or RNA, and no monoclonal antibody is available for specific binding to these bacteria. To make it worse, no PCR cycler is available for your project. Outline your experimental strategy to develop a novel method for detection of this bacterial species