Analytical detection limit of the PathoProofâ¢ Mastitis PCR Assay determined using two different experimental approaches

M.T. Koskinen; J. Holopainen; L. Salmikivi; H. Lehmusto; S. Niskala; J. Kurkela

Analytical detection limit of the PathoProofâ¢ Mastitis PCR Assay determined using two different experimental approaches

In: Mastitis control

Authors:

M.T. Koskinen

M.T. Koskinen Finnzymes Oy, Keilaranta 16A, 02150 Espoo, Finland Corresponding author: mikko.koskinen@finnzymes.fi

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J. Holopainen

J. Holopainen Finnzymes Oy, Keilaranta 16A, 02150 Espoo, Finland Corresponding author: mikko.koskinen@finnzymes.fi

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L. Salmikivi

L. Salmikivi Finnzymes Oy, Keilaranta 16A, 02150 Espoo, Finland Corresponding author: mikko.koskinen@finnzymes.fi

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H. Lehmusto

H. Lehmusto Finnzymes Oy, Keilaranta 16A, 02150 Espoo, Finland Corresponding author: mikko.koskinen@finnzymes.fi

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S. Niskala

S. Niskala Finnzymes Oy, Keilaranta 16A, 02150 Espoo, Finland Corresponding author: mikko.koskinen@finnzymes.fi

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J. Kurkela

J. Kurkela Finnzymes Oy, Keilaranta 16A, 02150 Espoo, Finland Corresponding author: mikko.koskinen@finnzymes.fi

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Type:: Chapter

Pages:: 183â189

DOI:: https://doi.org/10.3920/9789086866496_038

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The PathoProof Mastitis PCR Assay is a real-time PCR based reagent kit for identification of all major mastitis-causing pathogens. Here, we report on the analytical bacterial detection limit of the assay using two different experimental approaches. We first used purified DNA to prepare a standard dilution series from 450 to less than one genome copy of the assayâs bacterial targets and determined how many copies of each target could be detected by the test with >95% probability. In our second experiment, we measured sensitivity of the entire assay, including the DNA extraction procedure from raw milk samples. For this experiment, ten mastitis milk samples, shown to be negative for the tested targets, were randomly chosen, spiked with known quantities of the target bacterial colony forming units (cfu) and analysed using the assay. When sensitivity was measured based on the dilution series of purified DNA, the analytical detection limit of the test was as low as 5.6 bacterial genomes per PCR reaction. When using mastitis milk samples spiked with the bacterial targets, the detection limit was as low as <200 cfuâs per one milliliter of milk. Both experiments showed much variation in the detection limit of the test between the different bacterial targets. In conclusion, the results of this study demonstrate that the PathoProof Mastitis PCR Assay can detect low quantities of bacterial DNA, which is important for the mastitis testing laboratories now complementing or replacing bacterial culture based testing schemes with the assay. Variation in the detection limit between the different bacterial targets is explained by a combination of the biological characteristics of the bacteria and details related to the molecular specifications of the assay. This presentation discusses these variables as well as some of the challenges involved in detection limit validation of a PCR test in a controlled manner when using raw bovine milk as a sample matrix.

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Mastitis control

From science to practice

E-Book ISBN:: 9789086866496

Publisher:: Wageningen Academic

Print Publication Date:: 30 Oct 2008

Subjects
- Life Sciences

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Analytical detection limit of the PathoProofâ¢ Mastitis PCR Assay determined using two different experimental approaches

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Mastitis control

From science to practice

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Analytical detection limit of the PathoProofâ¢ Mastitis PCR Assay determined using two different experimental approaches