Comparison of fecal pooling methods and DNA extraction kits for the detection of Mycobacterium avium subspecies paratuberculosis
| Autor: | Tomoko Tasaki, Yasuyuki Mori, Katsuo Utiyama, Akiko Mita, Hitomi Mori, Tetsuo Nakagawa |
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| Rok vydání: | 2015 |
| Předmět: |
DNA
Bacterial Veterinary medicine Paratuberculosis Beef cattle Real-Time Polymerase Chain Reaction Sensitivity and Specificity Microbiology law.invention Feces law pooled fecal samples medicine Animals screening test Polymerase chain reaction Original Research biology business.industry biology.organism_classification medicine.disease Mycobacterium avium subspecies paratuberculosis DNA extraction Biotechnology Mycobacterium avium subsp. paratuberculosis Real-time polymerase chain reaction Herd Cattle Female Reagent Kits Diagnostic Commercial DNA extraction kits real‐time PCR business IS900 |
| Zdroj: | MicrobiologyOpen |
| ISSN: | 2045-8827 |
| DOI: | 10.1002/mbo3.318 |
| Popis: | The aim of the study was to develop a sensitive method using quantitative real‐time polymerase chain reaction (qPCR) with pooled fecal samples for the screening of Johne's disease (JD). Manufacturer‐specified and our new pooling method in combination with five commercial kits for DNA extraction and purification were compared. Different volumes of pooled fecal suspensions were tested, and the results were compared for individual samples and three pool sizes (5, 10, and 50 samples); each of the fecal suspensions, which were prepared from healthy dairy and beef cattle was spiked with 0, 10, 100, or 1000 cultured Mycobacterium avium subspecies paratuberculosis (MAP) organisms or was mixed with fecal suspensions from experimentally infected cattle. The MAP DNA detection proportion with our pooling method in combination with Johne‐Spin kit (Fasmac, Japan) was 100% for all models and all pool sizes, except for the low shedder model with a pool size of 50. There was no loss of sensitivity in pools of 10 subjects or less by using the new method. These results suggest that new method is a sensitive, practical, and cost‐effective screening test for the detection of MAP‐infected cattle and the monitoring of JD‐free herds. |
| Databáze: | OpenAIRE |
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