We compared ileal quantification outcomes produced by real-time PCR and culture-based
May 8, 2017
We compared ileal quantification outcomes produced by real-time PCR and culture-based methods in broiler chickens in a challenge model of necrotic enteritis. broiler market (5). Bacteria in the gastrointestinal tracts of chickens play an important role in health (15). Consequently quantification of the major bacterial areas in the chicken gut is essential for monitoring changes in microbial ecology in experiments involving challenge. Traditionally analysis of gastrointestinal areas offers depended on bacterial BMS-794833 culture-based counting methods or microscopy. The methodologies involved are time-consuming and require researchers to possess substantial microbiological experience. Furthermore potential bias is present since only those bacteria whose physiological and metabolic requirements are reproducible BMS-794833 can be cultivated (25). Molecular methods have been applied to quick characterization of bacteria. These methods include denaturing gradient gel electrophoresis (DGGE) (10 24 temp gradient gel electrophoresis (TGGE) (29) standard PCR (13) and terminal restriction fragment size polymorphism (T-RFLP) (23). These methods are able to determine relevant bacterial organizations BMS-794833 but the drawback of these techniques is that they are not fully quantitative and so cannot act as a stand-alone alternative method to culture and enumeration. In contrast real-time PCR can be used to quantify bacteria as the number of target gene copies can be determined in DNA extracted from samples. Hence real-time PCR has recently been used to enumerate bacteria in environmental samples (3 21 and in animal gastrointestinal tracts and feces (6 7 9 26 This study compared the precision of real-time PCR quantification of (PCR targeting the 16S rRNA genes of in the intestinal tracts of chickens in a challenge model of NE disease. Sampling of ileal digesta of the birds. The animal experiment was conducted as described recently (28). Briefly 1 350 birds were raised for 5 weeks with the birds in each Rabbit Polyclonal to MAP3KL4. cage assigned to one of nine treatment groups with six birds per treatment (25 birds/cage). On day 9 the birds in groups that would be challenged were given three species (Bioproperties Pty Ltd. Glenorie New South Wales Australia) and on days 14 15 and 16 they were inoculated with approximately 108 CFU of a pathogenic strain of type A (CSIRO Livestock Industries Geelong Victoria Australia). The BMS-794833 experimental design and the treatment acronyms are shown in Table ?Table1.1. On days 13 and 17 2 birds were randomly chosen in each cage and sacrificed for sample collections. Approximately 1 g of the ileal digesta was collected for microbial culture and a section of approximately 3 cm of ileum BMS-794833 (including digesta) was taken at the midpoint between Meckel’s diverticulum and cecal tonsils per bird for quantitative PCR analysis of infection and challengeusing the culture-based method followed the protocol described earlier (28). The bacteria were cultured and counted on Perfringens tryptose-sulfite-cycloserine and Shahidi-Ferguson Perfringens agar base mixed with egg yolk emulsion and Perfringens selective supplement (Oxoid). plates were incubated anaerobically for 48 h at 39°C prior to counting. Bacterial numbers were indicated as log10 CFU/gram of digesta. Removal of DNA from ileal content material was conducted utilizing a QIAamp BMS-794833 DNA feces package (Qiagen Hilden Germany) following a instructions of the maker with slight adjustments. Initial 180 to 220 mg freezing digesta was extracted from kept samples and cup beads (300 mg) (0.1 mm; Biospec Items Bartlesville Alright) were utilized to disrupt the cells in 400 μl of ASL lysis buffer by shaking the test on the miniBeadBeater (Biospec Items Bartlesville Alright) for 30 s. The cells had been after that lysed after adding 1 ml of ASL lysis buffer stool contaminants were eliminated and PCR inhibitors in the supernatant had been absorbed from the InhibitEX tablet. DNA was precipitated with the addition of 200 μl of ethanol captured for the QIAamp spin column cleaned by 500 μl of cleaning buffers AW1 and AW2 and eluted in 50 μl of TE buffer (10 mM Tris-HCl 1 mM EDTA [pH 8]). The quantitative real-time PCR assay was carried out by the technique of Smart and Siragusa (27). TaqMan common PCR master blend (Applied Biosystems Foster Town CA) was utilized. A set of primers (CPerf165F [5′-CGCATAACGTTGAAAGATGG-3′] and.