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ARS Home » Northeast Area » Frederick, Maryland » Foreign Disease-Weed Science Research » Research » Publications at this Location » Publication #153458
Title: AMPLI-DISK PCR, A NOVEL TECHNIQUE FOR ULTRA-SENSITIVE DETECTION OF BACTERIA

Author
item Schaad, Norman
item RANDHAWA, PARMJIT - CAL. SEED & PLANT LAB

Submitted to: Patent Application
Publication Type: Other
Publication Acceptance Date: 6/10/2003
Publication Date: 10/22/2003
Citation: Schaad, N.W., Randhawa, P. 2003. Ampli-disk PCR, a novel technique for ultra-sensitive detection of bacteria. Patent Application. DN 0110.03, SN 10/689,624.

Interpretive Summary: The invention describes a novel test element composed of an absorbent material (referred to as 'Ampli-disc') such as heavy-duty filter paper containing a selective agar medium for use in a polymerase chain reaction-based assay for ultra-sensitive detection of bacteria. The Ampli-discs are prepared by adding hot agar media and allowed to dry. The discs can be stored dry at room temperature until needed. Up to 10 ml of a sample, such as a seed wash, can be added to a single disc. After incubating the hydrated discs for 24-48 hours to allow the bacteria to grow, bacteria are washed from the disc and 1 microliter used for PCR. Results show a 10-100-fold increase in sensitivity over standard PCR techniques.

Technical Abstract: This invention describes a novel dried paper nutrient medium disk (Ampli-disk) for use in an ultra-sensitive PCR-based protocol for detection of bacteria. Ampli-disks are prepared by adding 4 ml of a hot semi-selective agar medium (0.5% agar) to a sterile 85-mm diameter heavy duty germination paper in a Petri plate, after drying in an oven for 1 hr at 50C, the dried disks are stored at 4C until needed. In this invention, 5 ml of an extract from seeds or other plant material is added to each of two Ampli-disks in separate Petri plates. The disks are incubated at 27C for 48 hrs to allow for growth of the target bacterium among the fibers of the Ampli-disk. For faster growing bacteria shorter times can be used. To extract the bacteria from the Ampli-disk, 5 ml of water is added and the disk incubated on a shaker at 100 rpm for 10 minutes. One ml of the extract is removed and archived at -20 for direct PCR and DNA extraction.