Development of a multiplex PCR for detection of Fusobacterium necrophorum, Streptococcus pyogenes and Streptococcus dysgalactiae subspecies equisimilis

Abstract

Streptococcus pyogenes, or group A streptococci (GAS) is a well known agent of pharyngitis. The same goes for the closely related S. dysgalactiae subspecies equisimilis (SDSE).
Recent data indicates that a third pathogen called Fusobacterium necrophorum, with similarly severe complications to GAS is the second most common pathogen in pharyngitis.
These infections cause the greatest damage in low and middle income countries, such as The Gambia in western Africa. Thus, there is currently a study to supplement ongoing studies of pharyngitis. To assist in the study and determine the proportions of F. necrophorum driven pharyngitis in The Gambia, the aim of this project was to develop a triplex assay for detection of GAS, SDSE and F. necrophorum. To accomplish this, 10 primer candidates were tested for specificity and optimal annealing temperature. The three pairs with highest performance were then assembled into a triplex assay and further optimized by adjusting primer concentration and probe concentration. The multiplex and the singleplex parts were then evaluated by real-time PCR of clinical samples already diagnosed for GAS, SDSE and/or F. necrophorum. Finally, the assays were tested in The Gambia on samples collected from patients with self reported pharyngitis. This led to the discovery of the first F. necrophorum induced pharyngitis in Gambia to our knowledge.

In the end, the new triplex proved to be robust with a high level of agreement with the already established clinical assays and with minimal performance loss from the transition from singleplex to multiplex assay. While efficiency can still be improved, the aim of creating a multiplex PCR against GAS, SDSE and F. necrophorum can be considered a success.

Popular Abstract

Making it multiplex

Have you ever had a sore throat? If yes, it was probably unpleasant. But for some people, it can even be deadly. One of the most common bacteria in pharyngitis are group A streptococci (GAS). In poorer countries such as The Gambia, GAS infections cause as many mortalities as malaria, which is why many studies are needed on the subject, especially in lower income countries. However, new data has shown that there might be another overlooked bacterium, that is just as severe as GAS. This bacterium is called Fusobacterium necrophorum. To investigate the matter, I created a multiplex PCR assay against GAS, F. necrophorum, and streptococcus dysgalactiae subspecies equismimilis (SDSE) which is a species closely related to GAS that can also cause serious infection.

So, what is a multiplex PCR? In a normal PCR, we have a pair of primers that bind to a DNA sequence to multiply it. That is a singleplex PCR. In a multiplex PCR, there are several primers. In my case, I need three to target three different bacteria. The beauty of PCR lies in its ability to quickly multiply even miniscule amounts of DNA into easily detectable levels. As I used a real-time PCR machine, I was able to view the DNA amplify in real time. To start off, I began gathering primers. I got 4 for GAS, 3 for SDSE and 3 for F. necrophorum. These were tested for 2 parameters: sensitivity and specificity. The primers should be sensitive and able to detect a low number of bacteria, but they should also detect only the bacteria I am looking for.

After getting rid of the worst performing primers, I had three primers left. After a little tweaking of the primer concentrations, it was time for the big test: Putting together the multiplex PCR and using it on samples that had already been tested by the clinic. Additionally, I ran the singleplex versions of the primers in my new multiplex to see if putting them together reduced their performances. The verdict after running 168 samples in total? Not bad. Compared to the singleplex there was no loss of performance (at least not any statistically significant loss) and compared to the clinic’s results my values were mostly similar with just a couple of differences.

Thus, I consider the multiplex to be fully functioning and ready to be deployed in The Gambia, where thousands of throat samples have been collected for a study of GAS infections. Maybe my multiplex will be used for a groundbreaking discovery? It remains to be seen.


Master’s Degree Project in Molecular Biology 30 credits 2024
Department of Biology, Lund University
Advisor: Lisa Wasserstrom
Advisors Unit/Department of translational medicine, Lund university
Co-advisor: David Nygren
Advisors Unit/Department of infectious disease, Lund university

• author: Borgström, Martin
• supervisor: Lisa Wasserström; David Nygren
• organization: Degree Projects in Molecular Biology
• course: MOBM02 20241
• year: 2024
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9176967
• date added to LUP: 2024-10-23 12:06:49
• date last changed: 2024-10-23 12:11:58