Lyophilisation improves the extraction of PCR‐quality community DNA from pig faecal samples

Ruíz, Raquel; Rubio, Luis A.

doi:10.1002/jsfa.3465

Cited by 25 publications

(29 citation statements)

References 27 publications

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“…Consequently, the test sensitivity was 10-fold greater than that reported previously (17,7). Lyophilization of feces has been reported to be useful for PCR-based studies of pigs (14), and our results indicate a useful role for the quantification of E. coli O157:H7 bacteria in cattle feces. Indeed, the slopes and the linear regression coefficients for the qPCR signal (C T values) and the known concentrations of microbial pathogen cells in the feces are in agreement with published values (2).…”

supporting

confidence: 38%

Lyophilization Prior to Direct DNA Extraction from Bovine Feces Improves the Quantification of Escherichia coli O157:H7 and Campylobacter jejuni

Rapp

Waller

Brightwell

et al. 2010

Appl Environ Microbiol

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Lyophilization was used to concentrate bovine feces prior to DNA extraction and analysis using real-time PCR. Lyophilization significantly improved the sensitivity of detection compared to that in fresh feces and was associated with reliable quantification of both Escherichia coli O157:H7 and Campylobacter jejuni bacteria present in feces at concentrations ranging between 2 log 10 and 6 log 10 CFU g ؊1

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supporting

confidence: 38%

Lyophilization Prior to Direct DNA Extraction from Bovine Feces Improves the Quantification of Escherichia coli O157:H7 and Campylobacter jejuni

Rapp

Waller

Brightwell

et al. 2010

Appl Environ Microbiol

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“…The pH was immediately measured in the crop content of each bird by using a Crison pH meter (Crison Instruments SA, Alella, Spain). Immediately after killing, contents from the crop, ileum (considered as the section between the Meckel's diverticulum and the ileo-cecal junction) and caeca of each bird were collected into plastic tubes, stored at −20°C and freeze-dried (Ruiz and Rubio, 2009). Samples of about 1 cm taken at the mid-point of the ileum of three randomly selected birds from each treatment were removed for histological analysis.…”

Section: Additivesmentioning

confidence: 99%

“…Eluted DNA was treated with RNase and the DNA concentration assessed spectrophotometrically by using a NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Purified DNA samples were stored at −20°C until use (Ruiz and Rubio, 2009). Bacterial log 10 number of copies was determined in faecal samples by using q-PCR.…”

Section: Additivesmentioning

confidence: 99%

Effects of inulin and di-d-fructose dianhydride-enriched caramels on intestinal microbiota composition and performance of broiler chickens

Peinado¹,

Echávarri²,

Ruíz³

et al. 2013

Animal

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In vitro and in vivo experiments were designed to evaluate the effectiveness of laboratory-made di-D-fructose dianhydride (DFA)-enriched caramels. The DFA-enriched caramels were obtained from D-fructose (FC), D-fructose and sucrose (FSC), or D-fructose and β-cyclodextrin (FCDC). In the in vitro experiment, raftilose and all caramels increased (P < 0.05) L-lactate concentration and decreased (P < 0.05) pH. Total short-chain fatty acid concentration was higher (P < 0.05) than controls in tubes containing raftilose, FSC, FCDC and commercial sucrose caramel (CSC). Raftilose, and all caramels tested except FSC and FC (1%), increased (P < 0.01) lactobacilli log 10 number of copies compared with the non-additive control. FSC, FCDC and CSC increased (P < 0.01) the bifidobacteria number of copies as compared with controls. All additives, except FCDC, decreased (P < 0.01) Clostridium coccoides/ Eubacterium rectale log number of copies. Compared with controls, raftilose, FC and CSC led to lower (P < 0.01) EscherichiaShigella and enterobacteria. For the in vivo experiment, a total of 144 male 1-day-old broiler chickens of the Cobb strain were randomly assigned to one of the three dietary treatments for 21 days. Dietary treatments were control (commercial diet with no additive), inulin (20 g inulin/kg diet) and FC (20 g FC/kg diet). Final BW of birds fed FC diet was higher (P < 0.01) than controls or inulin-fed birds, although feed: gain values were not different. Feed intake of chickens fed FC was higher (P < 0.01) than that of inulin-fed birds but not statistically different from controls. Crop pH values were lower (P < 0.01) in birds fed FC diet as compared with control diet, with inulin-fed chickens showing values not different from control-or FC-fed birds. Lower (P < 0.05) lactobacilli number of copies was determined in the crop, ileum and caeca of birds fed the inulin diet compared with the control diet. Inulin supplementation also resulted in lower (P < 0.05) C. coccoides/E. rectale, bacteroides and total bacteria in caecal contents. Addition of FC to broiler diets gave place to lower (P < 0.05) enterobacteria and Escherichia-Shigella in crop and caecal contents compared with controls. The bacteroides number of copies increased (P < 0.05) as compared with controls in the ileum, but decreased (P < 0.05) in the caeca of chickens fed the FC diet. Energy, ADF, NDF and non-starch polysaccharides faecal digestibilities were greater (P < 0.05) than controls in chickens fed diets containing inulin or FC. Fat digestibility was higher (P < 0.05) in FC-fed birds compared with controls or inulin-fed chickens. In conclusion, DFA-enriched caramels tested here, particularly FC, may represent a type of new additives useful in poultry production.

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“…Samples of rumen contents were freeze-dried before DNA extraction. Freeze-drying improves both the yield and quality of DNA extracted from gut contents (42). Freeze-dried samples were thoroughly mixed by physical disruption using a bead beater (Mini-Bead Beater; Biospec Products, Bartlesville, OK).…”

Section: Animals and Dietsmentioning

confidence: 99%

Bacterial and Protozoal Communities and Fatty Acid Profile in the Rumen of Sheep Fed a Diet Containing Added Tannins

Vasta

Yáñez-Ruíz

Mele³

et al. 2010

Appl Environ Microbiol

164

139

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This study evaluated the effects of tannins on ruminal biohydrogenation (BH) due to shifts in the ruminal microbial environment in sheep. Thirteen lambs (45 days of age) were assigned to two dietary treatments: seven lambs were fed a barley-based concentrate (control group) while the other six lambs received the same concentrate with supplemental quebracho tannins (9.57% of dry matter). At 122 days of age, the lambs were slaughtered, and the ruminal contents were subjected to fatty acid analysis and sampled to quantify populations of Butyrivibrio fibrisolvens, which converts C 18:2 c9-c12 (linoleic acid [LA]) to C 18:2 c9-t11 (rumenic acid [RA]) and then RA to C 18:1 t11 (vaccenic acid [VA]); we also sampled for Butyrivibrio proteoclasticus, which converts VA to C 18:0 (stearic acid [SA]). Tannins increased (P < 0.005) VA in the rumen compared to the tannin-free diet. The concentration of SA was not affected by tannins. The SA/VA ratio was lower (P < 0.005) for the tannin-fed lambs than for the controls, suggesting that the last step of the BH process was inhibited by tannins. The B. proteoclasticus population was lower (؊30.6%; P < 0.1), and B. fibrisolvens and protozoan populations were higher (؉107% and ؉56.1%, respectively; P < 0.05) in the rumen of lambs fed the tanninsupplemented diet than in controls. These results suggest that quebracho tannins altered BH by changing ruminal microbial populations.The fatty acid profile of the meat and milk of ruminants is strongly affected by diet (2, 15). When ingested, the dietary polyunsaturated fatty acids (PUFA) undergo a process known as biohydrogenation (BH) carried out by ruminal microorganisms (20). During the BH of C 18:2 (n-6) (linoleic acid [LA]) and C 18:3 (n-3) (linolenic acid [LNA]) a number of C 18:1 and C 18:2 isomers are formed (6). The last step in the BH process leads to the formation of C 18:0 (stearic acid [SA]). Among the intermediate products formed during this process, the isomer C 18:2 c9t11 (rumenic acid [RA]) is active in preventing cancer in mammals (17). Only a small amount of the RA found in meat and milk originates during BH. It is produced to a larger extent in muscle and mammary glands from the desaturation of Devillard et al. (11) have reported that protozoa do not have the capability of hydrogenating LA. The proportion of BH intermediates in the rumen can vary depending on changes in ruminal microbial populations (7, 51). Changes in ruminal fatty acid profiles are also reflected in intramuscular fatty acid composition (48,52).Tannins are phenolic compounds that are widespread in plants. When ingested by ruminants in large amounts, tannins can reduce the activity and the proliferation of ruminal microorganisms (34). Tannins from Lotus corniculatus (33) or from Acacia spp. (12) reduce the proliferation of B. proteoclasticus B316 T and B. proteoclasticus P18, respectively. Durmic et al. (12) reported that VA increased and SA decreased when extracts from Acacia iteaphylla, which contains condensed tannins (1), were incubated in vitro w...

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Lyophilisation improves the extraction of PCR‐quality community DNA from pig faecal samples

Cited by 25 publications

References 27 publications

Lyophilization Prior to Direct DNA Extraction from Bovine Feces Improves the Quantification of Escherichia coli O157:H7 and Campylobacter jejuni

Lyophilization Prior to Direct DNA Extraction from Bovine Feces Improves the Quantification of Escherichia coli O157:H7 and Campylobacter jejuni

Effects of inulin and di-d-fructose dianhydride-enriched caramels on intestinal microbiota composition and performance of broiler chickens

Bacterial and Protozoal Communities and Fatty Acid Profile in the Rumen of Sheep Fed a Diet Containing Added Tannins

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