Development of a polymerase chain reaction assay for quantification of Lawsonia intracellularis

Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
Suphot WattanaphansakRandall S Singer

Abstract

The objective of the present study was to develop a quantitative polymerase chain reaction (qPCR) assay using SYBR Green for quantification of Lawsonia intracellularis in cell culture and pig fecal samples. Specific primers were designed and tested using the aspartate ammonia-lyase (aspA) gene as a target. Serial 10-fold dilutions of cell culture samples and several sets of spiked feces were used for qPCR optimization. The lower limit of the linear range of the assay in cell culture was 5.1 x 10(2) L. intracellularis/ml. A concentration of between 2.55 x 10(4) and 2.55 x 10(3) L. intracellularis/g was the lower limit of the linear range when testing community DNA from spiked fecal samples. From both cell culture and fecal samples, L. intracellularis could be detected but not accurately quantified at levels approximately 1 log below the linear range. No cross-reactivity of qPCR was found when the assay was tested using the DNA extracted from 16 species of enteric bacteria commonly found in pig feces or closely related to L. intracellularis. The new qPCR assay might prove to be a sensitive, specific, precise, and accurate method for the detection and quantification of L. intracellularis in field samples.

References

Aug 1, 1994·Veterinary Microbiology·S McOristG H Lawson
Apr 1, 1996·Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc·P K HolyoakeM P Murtaugh
Feb 24, 2000·Journal of Comparative Pathology·G H Lawson, C J Gebhart
Mar 7, 2002·Journal of Clinical Microbiology·R H LindecronaK Møller
Oct 1, 2008·Veterinary Microbiology·Suphot WattanaphansakConnie J Gebhart
Jun 25, 2009·Journal of Applied Microbiology·H NathuesE grosse Beilage

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Citations

Jan 6, 2012·Journal of Clinical Microbiology·Fabio A VannucciConnie J Gebhart
Mar 19, 2014·Journal of Veterinary Pharmacology and Therapeutics·F SampieriD L Hamilton
Aug 4, 2016·Journal of Wildlife Diseases·Md Mukter HossainHo-Seong Cho
Jan 11, 2019·Equine Veterinary Journal·C E R PereiraC J Gebhart
Jan 17, 2020·Journal of Animal Science·Talita Pilar ResendeConnie Gebhart
Aug 1, 2020·PloS One·Carlos Eduardo Real PereiraRoberto Mauricio Carvalho Guedes

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Methods Mentioned

BETA
PCR
amplicon dissociation

Software Mentioned

Mx3000P
Basic Local Alignment Search Tool ( BLAST
Primer
MxPro

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