VALIDATION OF A REAL-TIME PCR ASSAY TO DETECT SALMONELLA IN CLINICAL SAMPLES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0209666
• Project Start Date: Oct 1, 2006
• Project End Date: Sep 30, 2007
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF PENNSYLVANIA
(N/A)
PHILADELPHIA,PA 19104

Performing Department
SCHOOL OF VETERINARY MEDICINE

Non Technical Summary
The rapid diagnosis of Salmonella is essential. To validate a real-time PCR assay to detect Salmonella

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3999	1103	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3999 - Animal research, general;

Field Of Science
1103 - Other microbiology;

Keywords

pcr

salmonella

sensitivity

Goals / Objectives
We propose to develop a diagnostic PCR assay to detect Salmonella, with a high sensitivity value.

Project Methods
The real-time PCR will be performed on a SmartCycler (Cepheid, Sunnyvale, CA) using PCR for the amplification of Salmonella DNA and a fluorogenic target-specific hybridization probe for the detection of the amplified DNA (Hoorfar et al, 2000). Details of primer and probe sequences are shown in Table 3. The invA probe was modified and was labeled at the 3 end with a black hole quencher rather than TAMRA. A 5ul aliquot of DNA is added to the PCR reagents which contain the Salmonella-specific primers and probe. The assay will also include internal control (IC) DNA and IC-specific primers and probe. The purpose of an IC is to detect specimens that may contain PCR inhibitors and it also helps to confirm the integrity of the assay reagents or malfunction of the thermal cycler (Shoeder et al,2003). Samples in which the IC is negative must be discarded and re-run.

Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: Fecal samples (496 from 218 equine patients) were subject to ten procedures for Salmonella detection. Three PCRs (invA gene, ttr gene) performed on unenriched feces and following overnight enrichment in either buffered peptone water (BPW) or tetrathionate (TTb). Four culture methods were also used; direct, following overnight enrichment in TTb or selenite, or a modified International Standards Organization (ISO) method. Test characteristics were calculated using standard techniques. Real-time PCR is useful for Salmonella detection in equine colic patients. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Fecal samples require overnight enrichment to yield consistent PCR results. Agreement between PCRs performed on enriched samples was excellent (kappa >0.81). Detection rates for Salmonella were poor by direct culture and following selenite or TTb enrichment. ISO was the most sensitive culture technique (70/496 positive). With ISO as "gold-standard", PCR (Se 52-60%, kappa 0.63-0.69) was more sensitive than culture from selenite (Se 31.4%, kappa 0.44) or TTb (Se 30.8%, kappa 0.42). Specificity for all methods was >99%. PCR offers better sensitivity than standard culture methods and results are available in 18-24 hours, compared to at least 3-5 days for ISO.

Publications

• Aceto H, Dallap B, OShea K, Graves AJ, Morrow JK, Rankin SC. Validation of Real-Time PCR for Targeted Surveillance of Salmonella in Equine Colic Patients. 2008 AAVLD Annual Meeting,Greensboro, NC.