PCR primers...a primer!

This post has been moved to the new Virology Down Under platform on Wordpress.

You can get to this specific post by clicking on the link below...

https://virologydownunder.com/pcr-primers-a-primer/

Please adjust your bookmarks.

Apologies for any inconvenience.

17 new MERS-CoV sequences bind perfectly to frontline screening PCR assay for MERS...

Click to enlarge. The primers/probe are depicted as grey boxes.
If mismatches existed they would show up as horizontal black
lines within the grey box. No mismatches are evident.
The GenBank accession numbers are
shown on the left of this alignment of 17 MERS-CoV
sequences.

Only 17 of the 45 sequences seem to include the region covered by the upE laboratory assay I just posted about in the WHO laboratory testing update but of those, the forward and reverse oligonucleotide primers and the probe all bind without any mismatch.

While that may sound like an obvious statement considering that these viruses were probably detected using that assay it isn't.

The new MERS-CoV sequences were determined using using unbiased 2nd generation high-throughput sequencing technologies that did not rely on these primers to generate them. So we are now able to check and see if there are any nucleotide changes at the target sites for the primers and probe, that would reduce the efficiency the assay.

There are no such oligonucleotide mismatches between primer and viral genes among those 17 sequences, which is good news for that assay's continued usefulness.

Built to last eh?

How reliable are the H7N9 real-time RT-PCRs for low viral loads?

An interesting document that compares the effectiveness of some different diagnostic PCR methods for use in H7 or N9-based flu diagnosis. It seems to show that in a couple of instances H7-specific or H9-specific molecular methods will fail (1:10,000,000 is a pretty extreme dilution) to detect H7N9 at low levels while some assay fail to amplify the intended target altogether. 

This sort of assay variation is pretty commonplace when you compare different PCR designs for the same target. It's a major reason why everyone prefers to design their own assay; each are convinced there's is the best. From this document, the people behind the OFFLU (FLI-H7) assay happen to be right. 

How these data relate to viral load in the human (and animal) samples these may be/have been used on is unknown. This sort of variance may contribute to negative throat swabs in H7N9-cases subsequently proven positive using lower respiratory tract samples (e.g. sputum) in which the viral load is presumed to be higher.

OFFLU: a network of expertise on animal influenza established jointly in 2005 by the World Organisation for Animal Health (OIE) and the Food and Agriculture Organization of the United Nations (FAO) to support and coordinate global efforts to prevent, detect and control important influenzas in animals.
CNIC: Chinese National Influenza Center

A(nother) new real-time PCR-based H7N9 test.

US CDC have made available their CDC H7N9 rRT-PCR for use on suspect cases (see interim guidance for defining cases) and in combination with their existing Flu rRT-PCR Dx Panel ("r"=real-time; "Dx"=Diagnosis/Diagnostic) after FDA support through an Emergency Use Authorization.