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PathoProof™ Mastitis PCR Assay
Frequently asked questions
Q 01: Who can use the PathoProof™ Mastitis PCR Assay?
The assay is intended for use in a laboratory. In other words, it is not a 'cow side' test. Any laboratory (for example bacterial culture laboratory or milk recording laboratory) can use the PathoProof Mastitis PCR Assay after product training. Installation of the assay and instrumentation involves a detailed product training for the laboratory staff.
Q 02: Do I need to have a molecular biology background to start using the PathoProof Mastitis PCR Assay?
The PathoProof assay is designed to be easy to use for any laboratory personnel, even if they have no molecular biology or PCR experience. The method is very straight forward and the results are automatically reported through the Norden Lab Mastitis Studio software.
Q 03: What laboratory equipment and reagents do I need for performing the PathoProof Mastitis PCR Assay?
The main piece of equipment needed is the Stratagene Mx3005P® QPCR System, supplied by Finnzymes. In addition, you will need some basic laboratory instrumentation like a microcentrifuge, vortex, incubator and pipettes. All of these are included in the PathoProof Mastitis PCR Assay Starter Pack available from Finnzymes.The PathoProof Mastitis PCR Assay includes all the necessary necessary reagents for DNA extraction and real-time PCR.
Q 04: What kind of training does a laboratory need before it can start using the assay?
After a laboratory has decided to start using the assay, our product specialist will provide detailed training for the laboratory staff. This is typically done in connection with installation of the real-time PCR instrument to the labratory. The training takes approximately 3-4 days and involves analyzing milk samples from start to finish multiple times to gain some routine, as well as detailed training of the real-time PCR instrument and the software used for interpretation of the results.
Q 05: How many samples per day can I handle with the PathoProof Mastitis PCR Assay?
One operator can test approximately 100–200 milk samples per day.
Q 06: How many samples can be tested with one real-time PCR instrument?
The capacity of one instrument is approximately 17 000 samples per year.
Q 07: What is the shelf life of the PathoProof Mastitis PCR Assay kits? Do I have to use the whole kit at once, or can I use it in portions?
The PathoProof Mastitis PCR Assay kits are stable for six months from the date of packaging when stored and handled properly. The reagents can be divided and used at several occasions. The F-870S kit is suitable for laboratories handling small sample numbers in each DNA extraction session. The F-870L kit is designed for laboratories performing DNA extraction for large sample numbers in each DNA extraction session.
Q 08: Which real-time PCR instruments can be used with the PathoProof Mastitis PCR Assay?
The Stratagene Mx3005P QPCR System should be the instrument of choice for use with the PathoProof Mastitis PCR Assay. The instrument should be purchased from Finnzymes directly. If a laboratory already has a real-time PCR instrument, the 7500 Fast Real-Time PCR System from Applied Biosystems and the Chromo4 Real-Time PCR Detection System from Bio-Rad Laboratories have also been validated for use with the assay.
Q 09: What is the sensitivity and specificity of the PathoProof Mastitis PCR Assay, and how does that compare to the sensitivity and specificity of bacterial culturing?
The analytical sensitivity and specificity (=analytical accuracy in identification of well-characterized bacterial strains) of the PathoProof Mastitis PCR Assay has been shown to be 100% for all bacteria identified by the test (Koskinen et al., 2009). This makes the assay the most accurate mastitis test ever developed.
Because the PathoProof Mastitis PCR Assay accurately detects bacterial DNA, whereas bacterial culturing identifies viable bacterial cells with varying accuracy depending on the operator, performance comparisons between the two methods are very challenging to conduct. If bacterial culturing is assumed to represent the 'golden standard', and performance of the PathoProof Mastitis PCR Assay is compared against it, the sensitivity of the assay has varied between ~90% and 100% depending on species and the type of milk sample.
Since the assay was made available, numerous scientific studies have been conducted on the subject. The studies have focused on performance comparisons from quarter milk samples from clinical and subclinical mastitis, as well as on comparing the sensitivity and specificity of the PathoProof Mastitis PCR Assay from metered milk samples against bacterial culturing from udder milk samples. These studies will appear in print in scientific literature during 2009 and 2010.
Q 10: Which bacterial species does the PathoProof Mastitis PCR Assay detect?
The assay detects all main mastitis-causing species and the β-lactamase penicillin resistance gene in staphylococci.
Click here to see the bacterial species and groups identified by the assay.
Large-scale prevalence studies indicate that >95 % of all clinical and subclinical mastitis cases are caused by these species.
Q 11: Does the PathoProof Mastitis PCR Assay detect Mycoplasma?
Yes. Mycoplasma bovis together with S. aureus and Str. agalactiae can be identified with PathoProof Mastitis Major-3 Kit in just four hours.
Q 12: Is the PathoProof Mastitis PCR Assay applicable to sheep milk?
The DNA extraction and real-time PCR reagents of the assay are not inhibited by sheep milk. However, specificity and sensitivity validation studies of the assay have not been performed with bacteria originating from caprine milk samples.
