Tag Archives: multiplex PCR

“On the Road….”

In Katoomba (Australia) at the moment for a virus conference.


Katoomba is rather a stunning place with outrageous views. I have done a couple of early morning runs along the clifftops, which have been amongst the best I have ever done anywhere in the world. I would recommend this place for a visit to anyone who is visiting the Sydney region.

And what about the virology..?

The talks so far have generally been very good, and I will detail a couple of them in the next few weeks, but I have “taken home” a couple of things from the conference to date.

  • Clinical Virology has come a long way in the past 20 years. From the number of viruses that can now be diagnosed in real-time, the number of viral vaccines available, and the range of viruses which can be treated with effective anti-virals… Things have really moved on fast since when I was at medical school in the early 90s when virology was a relatively small sub-specialty compared with bacteriology, with retrospective diagnoses and few anti-viral options. Now virology stands right alongside bacteriology and there is every possibility it could move on past it. Given this, I have made a mental note to try and get to a virology conference on an annual basis to keep up to date with progress.
  • In the research setting and in large clinical centres, multiplex viral assays are now commonplace for the laboratory diagnosis of viral infection in a number of different systems; respiratory, gastro-intestinal, and CSF being the predominant ones. These multi-plexes are not of exorbitant cost. However if samples from every patient presenting with an Upper Respiratory Tract Infection or  Gastroenteritis were tested with a viral multiplex PCR the additive cost may soon become prohibitive… I thus see the importance of laboratories of developing “testing criteria” for each of these viral multiplex PCRs so that they can be used on the patients who most need them and where they are going to make most difference to the eventual clinical outcome. I resolve to make this a focus of my work over the next couple of years.

Coffee break finished. Back to the conference…..



“PCR: A Multiplicity of Multiplexes….”

PCR multiplexes seem to be all the rage just now….

Here is just a selection of what is currently available in New Zealand:

  • CSF Multiplex: HSV, VZV, Enterovirus, Parechovirus, N. meningitidis, S. pneumoniae
  • Respiratory Virus Multiplex: Influenza A&B, CMV, Adenovirus, RSV, hMPV, Rhinovirus, Coronavirus, Parainfluenza 1-3, Bocavirus.
  • Atypical Pneumonia Multiplex: Legionella, Mycoplasma pneumoniae, Chlamydia, Pneumocystis, Bordetella pertussis & parapertussis.
  • Enteric Virus Multiplex: Norovirus, Rotavirus, Adenovirus, Astrovirus.

However there are some downsides to multiplex PCRs, both clinical and technical. These are as follows:

  •  Cost: The clinician may not want to test for all the assays within a multiplex PCR, therefore the cost may be more than with other individual assays that are required. For example, it is usually easy to differentiate between a viral and a bacterial meningitis based on initial CSF findings. However if the “CSF multiplex” includes both bacteria and viruses, then it may lead to unnecessary cost as well as problems with positive predictive value as described below.
  • Expertise: Carrying out a multiplex PCR still requires a reasonable amount of expertise, particularly if the reagents are being prepared “in-house” The expertise level increases further when troubleshooting is required.
  • Controls: Controlling each assay within the multiplex.
  • Test Volumes: Because of the amount of controls required per batch, significant numbers of tests are often required to make it cost-effective. Therefore may restrict some multiplexes to the larger centres.
  • Optimisation: Optimising each assay and avoiding competitive inhibition between the different reagents.
  • Positive predictive Value: If you have 5 tests in a multiplex PCR, then it is likely that at least one of these tests has a very low pre-test probability making interpretation of positive results difficult. For example, during the Influenza season, it may be prudent to test for Influenza first and then worry about other diagnoses if this test is negative.
  • More than one positive result: For example if you are doing a multiplex PCR with 7 or 8 respiratory viruses, it is not uncommon for 2 or even 3 assays to be positive. You then need to decide which one is causing the problem….
  • Only diagnoses what is tested for in the multiplex: I.e. It is not a catch-all method.

 Multiplex PCR can clearly be very useful in some situations. However it is important to be aware of the limitations as described above and have other testing options available. Otherwise the skill of utilising laboratory tests in a cost effective and clinically appropriate manner will be lost….


For a really simple walk through the basics of the PCR reaction, check out this website. I will go into a bit more detail next week on detection of PCR product, Real-Time PCR etc.