Real - time PCR Working Group
Tuesday 19th September 2012
Parklands Hotel, Perth
Present: Dave Yirrell, Kathleen Harvey-Wood, Susan McDonagh, Pauline Furness,Geraldine Kaminski, John Shone
Diagenode Representative: Jean-Marie Hoornaert
1.Apologies:Kate Templeton Roger Evans, Lesley Dargie
2.Minutes of last meeting: accepted.
3.WinterSeason(Respiratory and norovirus)
Dundee:Last year there was an agreementfor 6 day working over the winter which was neverincreased to 7 days as planned.A similar arrangement for the coming season is being negotiated. However, previous payments for staff are being replaced by Agenda for Change rates so staff may be less willing to take part. Obligatory six day working is likely to be instigated but this will be difficult to implement as we will be 2 staff down.
Southern General/ Yorkhill: There is still a BMS on site and on call for Yorkhill.
POCT used for RSV (Binax). If negative, samples are sent toGartnavel for a full screen.
Aberdeen:Six day working is currently in place.
Inverness: Testing has been expanded to include a larger respiratory panel, which is undergoing final validation. A 5 day service is in place with further arrangements to be negotiated. An on-call service is currently available for DIF testing of NPA samples. Norovirus testing by PCR has been set-up but current equipment limitations mean that faeces samples cannot be processed. Testing will be included when new equipment is in place.
4.Software
New version software (v2.06) for ABI has solved previous problems. It has a completely different format but is easy to use.
5.Equipment/Testing
Dundee: There is a plan being explored to get all faeces samples (13,000 p/a) tested by molecular systems. This will provide results for bacteria, viruses and some parasites within 24 hours. Negotiations are underway with commercial partners and business case being developed. Positives will be cultured for sensitivities and typing.
Southern General/ Yorkhill: The QIAsymphony + AS module + Rotor-gene add-ons in use. There has been a software problem with Artis V4 kits when extraction stage is not required (e.g. on previously extracted samples).
Aberdeen: QIAsymphony now in place for BBV testing with set-up platform as add-on.
Inverness: Awaiting easyMAG, additional ABI7500 and Cepheid (currently under test), but these have been held up by the MSC. It is not clear whether these will be on-site prior to the winter season.
Cepheid: False results for MRSA/MSSA were obtained on three occasions in Aberdeen. Indeterminate results have been obtained elsewhere (SM and PF to follow-up). It was also noted that this Flu assays on the system have not been sensitive. This may be in part due to the limited number of sample tests released by the company to carry out a full comparison.
6.Internal controls
Most labs are designing new assays with integral IC. The need for an IC in each test well was discussed. Most sites are currently using only one IC test per sample.
7.Staffing / Concerns
Staff: All sites are under pressure from decreased staff numbers and grade reduction. Most felt that although junior staff are capable of molecular testing, full training is essential. This is particularly important for staff with no or limited experience of such work. An example was given of work-up for bacterial assay which require single colonies for pure growth. In many cases complications had arisen from incorrect selection of colonies which resulted in mixed results. These problems are especially important with extended working day/ on call / winter season.
R&D: is severely limited or no longer possible at most sites (though no representation from Edinburgh and Glasgow) which is causing concern.
Validation and quality: discussion was around the need for validation if tests had been previously validated elsewhere using the same equipment and reagents. There is still a need for validation reports for all assays, but these can be minimal (testing a small panel of samples) if a full validation had been carried out elsewhere. It was noted that work-up and validation can be difficult with current pressures. Many labs are in a state of change and flux where even limited validation of commercial assays has been difficult
- Developments
See above. Development has not been possible on most sites due to limitations with staff/ time.
- Date of next meeting:
Tuesday 13thMarch 2013, Parklands Hotel, Perth.
Presentation by Jean-Marie Hoornaert, Diagenode
The Company
Based in Liege, Belgium with 60 staff.
Diagnostic kits have been targeted towards infectious diseases, but not “blockbusters” such as blood borne viruses. These are based on systems readily available in labs and they are also working with other companies to create assays using specific systems e.g.BDmax. Development includes full work-up using a range of systems and reagents so that kits are validated for a range of formats. All tests contain internal controls from the extraction stage on. Beta kits are then used for compatibility studies: extraction on easyMAG, MagNApure, QIAsymphony; amplification on ABI, iCycler, Rotor-gene, Smartcycler. Evaluation is carried out for sensitivity (ATCC controls), specificity, stability, reproducibility before final CE clearance. Thereafter they wish to work with partners to gain clinical validation of the assays.
Products
These are available as different combinations as panels. A RespEasy kit is about to be launched which contains reagents for 14 pathogens in a dry strip format with results available in 2 ½ hours after addition of mastermix and 5µl sample. DNA and RNA assays are carried out using identical conditions, so all assays can be run together. Prices range from €210 per 100 tests (single/duplex) to €50 each for the respiratory screen.
They are willing to prepare bespoke sets of reagents for specific customers. These can be freeze dried to ensure a long shelf life.
Gastro: C.diff, shigella, noro, rota, adeno, astro
Herpes: CMV (qualitative and quantitative using standard curve), HHV6, EBV, HS1, HS2, VZ
STD: Ct, GC, Mycoplasma, gardnerella, Tv
Meningitis: Neisseria A,B,C, Strep pn and others
Respiratory: FluA,B, PIV1-4, RSV, rhino, metapneumo, c. pneumonia, mycoplasma, legionella, M.tb, CAP
The scientific support and back-up appears to be an important part of this company. They are willing to supply test kits for labs to trial and welcome feedback. Contact details are:
Jean-Marie Hoornaert, Sales and Marketing Manager Diagnostic,
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