Pedersen Ken Steen, Pedersen Klaus H, Hjulsager Charlotte, Larsen Lars Erik, Ståhl Marie, Stege Helle, Angen Øystein, Nielsen Jens Peter
Department of Large Animal Sciences, University of Copenhagen, Groennegaardsvej 2, DK-1870 Frederiksberg C, Denmark.
J Vet Diagn Invest. 2012 Sep;24(5):968-70. doi: 10.1177/1040638712452111. Epub 2012 Jul 11.
Absolute quantification of Lawsonia intracellularis by real-time polymerase chain reaction (PCR) is now possible on a routine basis. Poor repeatability of quantification can result in disease status misclassification of individual pigs when a single fecal sample is obtained. The objective of the current study was to investigate overall variation within a day for fecal numbers of L. intracellularis bacteria determined by real-time PCR in growing pigs. From each of 30 pigs with an infection of L. intracellularis, 5 fecal samples were collected within 1 day. A total of 150 fecal samples were obtained and subjected to quantitative PCR (qPCR) testing. Mean fecal dry matter content was 14.3% (standard deviation = 4.5%). Two pigs (6.7%) alternated between being L. intracellularis qPCR positive and negative. For 28 pigs, the excreting levels of L. intracellularis were within the dynamic range of the qPCR assay at all sampling points. For these 28 pigs, the mean excretion level of L. intracellularis was 6.1 log(10) bacteria/g feces (standard deviation = 1.2 log(10) bacteria/g feces). The maximum observed difference between 2 fecal samples from the same pig was 1.1 log(10) bacteria/g feces. The average standard deviation for individual pigs was 0.27 log(10) bacteria/g feces. The average coefficient of variation within pigs was 0.04, ranging from 0.006 to 0.08. The results imply that absolute quantification of L. intracellularis by qPCR has acceptable repeatability within 1 day. However, a population-specific proportion of pigs alternating between positive and negative test results must be expected.
现在，通过实时聚合酶链反应（PCR）对胞内劳森菌进行绝对定量已成为常规操作。当仅采集单个粪便样本时，定量的重复性较差可能导致对个体猪的疾病状态误判。本研究的目的是调查生长猪中通过实时PCR测定的胞内劳森菌粪便数量在一天内的总体变化情况。从30头感染胞内劳森菌的猪中，每头猪在一天内采集5份粪便样本。共获得150份粪便样本并进行定量PCR（qPCR）检测。粪便平均干物质含量为14.3%（标准差 = 4.5%）。两头猪（6.7%）的胞内劳森菌qPCR检测结果呈阳性和阴性交替。对于28头猪，在所有采样点，胞内劳森菌的排泄水平均在qPCR检测的动态范围内。对于这28头猪，胞内劳森菌的平均排泄水平为6.1 log₁₀菌/克粪便（标准差 = 1.2 log₁₀菌/克粪便）。同一头猪的两份粪便样本之间观察到的最大差异为1.1 log₁₀菌/克粪便。个体猪的平均标准差为0.27 log₁₀菌/克粪便。猪体内的平均变异系数为0.04，范围为0.006至0.08。结果表明，通过qPCR对胞内劳森菌进行绝对定量在一天内具有可接受的重复性。然而，必须预期有一定比例的猪会出现检测结果在阳性和阴性之间交替的情况。
