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ARS Home » Northeast Area » Frederick, Maryland » Foreign Disease-Weed Science Research » Research » Publications at this Location » Publication #193651
Title: ASSESSMENT OF MULTIPLE DISPLACEMENT AMPLIFICATION FOR INCREASING PCR SENSITIVITY

Author
item Sechler, Aaron
item ZHAO, TINGCHANG - ACADEMY SCIENCE, CHINA
item Schaad, Norman
item Schuenzel, Erin

Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 3/22/2006
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Polymerase chain reaction (PCR) has become a standard tool in disease diagnosis. For an assay to be useful the primers and probe must be both specific and sensitive. Although some primers and probes are specific at the subspecies or strain level they are not sensitive enough to detect bacteria at levels lower than 1,000 colony forming units per ml. Recently, multiple displacement amplification (MDA) (Dean, F.B., 2002. Proc. Natl. Acad. Sci. 99-5261-66) has been employed for uniform amplification of whole genome DNA to increase the amount of DNA available for analysis of dried blood samples. For MDA of whole genome DNA, we used a REPLI-g® kit as described by the manufacture (Qiagen, Valencia, CA.). We compared direct real-time PCR to MDA-PCR for detection of two regulated pathogens, Xanthomonas campestris pv. campestris (Xcc) in crucifer seeds and Acidovorax avenae subsp. citrulii (Aac) in melon seeds. These bacteria were chosen because their real-time primers and probes had high specificities but low sensitivities. DNA extraction and MDA increased the sensitivity in 14/19 cases where Xcc and Aac were negative in the standard real-time PCR. In 3/19 samples, detection was accomplished with DNA extraction alone. This simple technique allows the use of primers and probes with high specificity but low sensitivity for detecting bacteria in low numbers in plant tissues.