Fu C J, Carter J N, Li Y, Porter J H, Kerley M S
Department of Animal Sciences, University of Missouri, Columbia, MO 65211, USA.
Lett Appl Microbiol. 2006 May;42(5):490-4. doi: 10.1111/j.1472-765X.2006.01893.x.
To compare agar plate and real-time PCR methods on enumeration of total anaerobic bacteria, Lactobacillus and Clostridium perfringens in dog faeces.
Thirty-two faecal specimens from Labrador retriever dogs were used to compare agar plate and real-time PCR enumeration methods for Lactobacillus, C. perfringens and total anaerobic bacteria. Total anaerobic bacteria, C. perfringens and Lactobacillus of faeces were counted (as CFU g(-1) faeces) for 48-h incubation at 37 degrees C in an anaerobic gas chamber on genus-selective media. Total genomic DNA from samples was extracted by the QIAamp DNA stool mini kit. The quantification of DNA (as DNA copy per gram faeces) by real-time PCR was performed with a LightCycler system with the QuantiTect SYBR green PCR kit for PCR amplification. The results indicated that there was a significant correlation between CFU and DNA copy of Lactobacillus (R2 = 0.78, P < 0.01) and total anaerobic bacteria (R2 = 0.21, P < 0.05); but no correlation was found between CFU and DNA copy of C. perfringens. The regression equations for Lactobacillus and total anaerobic bacteria were log(DNA copy) = 0.83 x log(CFU) + 1.43 and log(DNA copy) = 1.62 x log(CFU) - 6.32 respectively.
The real-time PCR method could be used to enumerate Lactobacillus within 2 days when compared with plating method which requires 5-6 days.
The real-time PCR method and the primer set for Lactobacillus spp. harboured in the dog intestine can be used for rapid enumeration of lactobacilli and monitoring of the faecal Lactobacillus community.
比较琼脂平板法和实时荧光定量PCR法对犬粪便中总厌氧菌、乳酸杆菌和产气荚膜梭菌的计数。
采用32份拉布拉多猎犬的粪便标本，比较琼脂平板法和实时荧光定量PCR法对乳酸杆菌、产气荚膜梭菌和总厌氧菌的计数。将粪便中的总厌氧菌、产气荚膜梭菌和乳酸杆菌在厌氧培养箱中于37℃在属选择性培养基上培养48小时后进行计数（以CFU g(-1)粪便计）。用QIAamp DNA粪便微量提取试剂盒从样本中提取总基因组DNA。使用LightCycler系统和QuantiTect SYBR绿色PCR试剂盒进行PCR扩增，通过实时荧光定量PCR对DNA进行定量（以每克粪便的DNA拷贝数计）。结果表明，乳酸杆菌的CFU与DNA拷贝数之间存在显著相关性（R2 = 0.78，P < 0.01），总厌氧菌的CFU与DNA拷贝数之间也存在显著相关性（R2 = 0.21，P < 0.05）；但产气荚膜梭菌的CFU与DNA拷贝数之间未发现相关性。乳酸杆菌和总厌氧菌的回归方程分别为log(DNA拷贝数) = 0.83 x log(CFU) + 1.43和log(DNA拷贝数) = 1.62 x log(CFU) - 6.32。
与需要5 - 6天的平板法相比，实时荧光定量PCR法可在2天内对乳酸杆菌进行计数。
实时荧光定量PCR法及用于犬肠道中乳酸杆菌属的引物组可用于快速计数乳酸杆菌并监测粪便中乳酸杆菌群落。
