Mitochondrial multiplex real-time PCR as a source tracking method in fecal-contaminated effluents

Multiplex real-time PCR amplifying fecal mitochondrial DNA (mtDNA) combined with rapid, crude DNA preparations are promising additions to surface water source tracking methods. Amplification of eukaryotic mitochondrial DNA identifies the fecal source directly and can be used in conjunction with other intestinal microbial methods to characterize effluents. Species-specific primers and dual-labeled probes for human, swine, and bovine NADH dehydrogenase subunit 5 (ND5) genes were created for multiplex real-time PCR in feces and effluent slurries. The linear range of the multiplex assay was 10(2)-10(7) mtDNA copies for human, bovine, and swine effluent in combination (equal volumes). PCR amplification efficiencies for bovine, human, and swine mtDNA when assayed in combination were 93, 107, and 92% respectively. Linear regression correlation coefficients (r2) were 0.99 for all standard curves except for human mtDNA in combination (r2 = 0.95). Multiplex amplification of bovine, human, and swine mtDNA (ND5) exhibited no cross-reactions between the effluents from three species of interest. Also, no cross-reactions were observed with effluents of other vertebrates: sheep, goat, horse, dog, cat, Canada goose, broiler, layer, turkey, and tilapia. Performed as a blind test, the PCR operator was able to correctly identify all but two effluent challenge samples (10/12 or 83% correct) with no false positives (22/22 or 100% correct). The multiplex assay had a tendency to detect the species of highest mtDNA concentration only. Better detection of all three species in a combination of human, bovine, and swine effluents was accomplished by running each real-time PCR primer/ probe set singly. Real-time PCR detection limit was calculated as 2.0 x 10(6) mitochondrial copies or 0.2 g of human feces per 100 mL effluent. Some carry-over mtDNA PCR signal from consumed beef, but not pork, was found in feces of human volunteers.     

Assessment of the relative sensitivity of milk ELISA for detection of Mycobacterium avium ssp. paratuberculosis infectious dairy cows

Milk ELISA are commonly used for detection of Mycobacterium avium ssp. paratuberculosis (MAP) antibodies in dairy cows, due to low cost and quick processing for large numbers of samples. However, low sensitivity and variations from host and environmental factors can impede detection of MAP antibodies at early disease stages. The objectives of our study were to assess the sensitivity of milk ELISA in comparison with fecal tests and to evaluate how detectable antibody concentrations in milk vary with changes in fecal shedding of MAP, cow age, cow parity, days in milk, and time of year. To compare the sensitivity of a commercial milk ELISA with solid and broth fecal culture and with fecal real-time PCR, a longitudinal study was performed for the identification of MAP-infectious animals as determined by prior fecal testing for MAP shedding. In addition, associations between variation in milk MAP ELISA score and changes in fecal MAP shedding, host age, days in milk, and season were evaluated. Monthly milk and fecal samples were collected over 1 yr from 46 cows that were previously shedding MAP in their feces. Sensitivity of milk ELISA was 29.9% (95% CI: 24.8 to 35.1%), compared with 46.7% (40.7 to 52.7%) for fecal solid culture, 55.0% (49.3 to 60.7%) for fecal broth culture, and 78.4% (73.3 to 83.1%) for fecal direct real-time PCR. The effect of stage of lactation could not be separated from the effect of season, with increased milk ELISA scores at greater days in milk in winter. However, unpredictable monthly variations in results were observed among the 3 assays for individual cow testing, which highlights the importance of identifying patterns in pathogen and antibody detection over time in MAP-positive herds.     

Application of host‐specific source‐tracking tools for rapid identification of fecal contamination in fresh produce by humans and livestock

BACKGROUND: Fecal contamination in fresh produce is a public health concern because it may contain human pathogens. We introduced host‐specific quantitative real‐time polymerase chain reaction (qPCR) assays for the rapid detection and identification of fecal contamination sources from humans and farm animals (cow, pig, chicken) in fresh produce. Each composite fecal sample was spiked on lettuce at two contamination levels (0.2 mg or 2 mg feces g^?1), followed by qPCR assays for detecting each host‐specific genetic marker: BoBac (cow); PF163 (pig); CP3‐49 (chicken); and HF183 and gyrB (human). Two commercial DNA extraction kits were compared to evaluate DNA recovery yields and removal of PCR inhibition. Sketa2 assay was conducted to assess the presence of PCR inhibition in the contaminated lettuce. RESULTS: All the qPCR assays yielded reliable detection from contaminated lettuce (2 mg feces g^?1), where their target gene numbers were 1.5–5.0 × 10³ (HF183), 0.8–2.2 × 10³ (gyrB), 0.6–1.6 × 10³ (BoBac), 1.6–3.0 × 10³ (CP3‐49) and 1.1–2.2 × 10³ (PF163) copies g^?1 of lettuce. Among the two extraction kits, QIAamp DNA Stool Kit resulted in 2–3 times higher sensitivity and 20% less PCR inhibition than the PowerFood^? kit. CONCLUSION: This study provides information on the optimized host‐specific qPCR assay in identifying sources of fecal contamination in fresh produce and is useful for tracking the contamination source and improving agricultural practice. © 2012 Society of Chemical Industry     

Rapid culture-independent quantitative detection of enterotoxigenic Escherichia coli in surface waters by real-time PCR with molecular beacon

Rapid and reliable detection of enterotoxigenic Escherichia coli (ETEC) is critical for the management of the waterborne diseases threatening human lives worldwide. In this study, a culture-independent real-time PCR assay, in molecular beacon format was designed and validated for detection and quantitative enumeration of ETEC harboring LT1 gene (encoding heat labile toxin) in surface waters contaminated by fecal pollutants of human and animal origin. It was observed that the assay was able to detect 2 CFU/mL of ETEC (r = 0.997; PCR efficiency = 99.8%) from water samples spiked by a reference organism (E. coli MTCC 723). In the presence of 10(6) CFU/mL of nonpathogenic E. coli(E. coli DH5alpha), the lowest detection limit from spiked water samples was 4 CFU/mL. The assay was 500 times more sensitive than conventional PCR using the same oligomers (Student's t test p < 0.05). The assay could specifically detect and quantify ETEC (1.2 x 10(3) to 1.4 x 10(6) CFU/100 mL) in polluted surface waters of river Gomti. The rapid culture-independent assay developed in this study for detection and quantitative enumeration of ETEC can be used for preliminary monitoring of surface waters to prevent waterborne outbreaks.     

Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR

A method combining immunomagnetic separation (IMS) and real-time (5'-nuclease) PCR was developed to detect Escherichia coli O157:H7. Monoclonal antibody specific for the E. coli O157 antigen was added to protein A-coated magnetic particles to create antibody-coated beads. The beads specifically captured E. coli O157:H7 from bacterial suspensions. The cells were eluted from the beads and lysed by heating; the eluate was then assayed by real-time PCR, using primers and probe specifically targeting the eaeA gene of E. coli O157:H7. Approximately 50% of the cells in suspension were captured by the beads and detected by real-time PCR. No cross-reactivity was detected when other strains of E. coli were tested. This method was applied to detect E. coli O157:H7 from ground beef. Both cell capture efficiency and real-time PCR efficiency were reduced by meat-associated inhibitors. However, we were still able to detect up to 8% of E. coli O157:H7 from inoculated ground beef samples. The detection sensitivity varied among ground beef samples. The minimum detection limit was <5x10(2) cells ml(-1) for suspensions of E. coli O157:H7 in buffer and 1.3x10(4) cells g(-1) for E. coli O157:H7 in ground beef. The combination of IMS and real-time PCR results in rapid, specific and quantitative detection of E. coli O157:H7 without the need for an enrichment culture step.     

Bifidobacterium species isolated from animal feces and from beef and pork meat

Bifidobacteria were isolated from 122 of 145 samples of animal feces (from cattle, swine, sheep, goats, horses, rabbits, chickens, geese, and pigeons) from farms in France and Austria and from 92 of 955 production and processing chain samples of beef and pork (obtained at slaughter, cutting, and retail). Bacterial strains were identified to species by phenotypic numerical classification based on API 50CH and ID 32A tests and DNA-DNA hybridization. Bifidobacterium pseudolongum was present in 81% (99 of 122 samples) of all Bifidobacterium-positive fecal samples and predominated in samples from all animal species except those from swine from Austria. In these Austrian swine samples, the majority of strains were identified as Bifidobacterium thermophilum (78%), followed by B. pseudolongum (48%). The distribution of B. thermophilum and B. pseudolongum differed significantly between Austrian swine and cattle samples such as those collected along beef and pork production and processing chains. Bifidobacterium animalis was isolated from swine feces, and Bifidobacterium ruminantium was isolated from cow dung. Six fecal isolates (from cattle, swine, rabbits, goats, and horses) were identified as belonging to Bifidobacterium species of predominantly human origin: B. adolescentis, B. bifidum, and B. catenulatum. Only one other species, Bifidobacterium choerinum, was detected with low frequency in a pork processing chain. B. pseudolongum subsp. pseudolongum was predominant in pig feces, whereas B. pseudolongum subsp. globosum was predominant in feces from other animal species. Four strains closely related to both subspecies (58 to 61% DNA reassociation) formed a distinct genomic group. PCR techniques, which are more rapid and sensitive than culture-based methods, could be used to detect directly B. pseudolongum and B. thermophilum as indicators of fecal contamination along the meat processing chain.     

Evaluation of immunomagnetic separation and PCR for the detection of Escherichia coli O157 in animal feces and meats

Series of animal feces and meat samples artificially contaminated with strains of Escherichia coli O157 isolated from different sources were tested by both an immunomagnetic separation (IMS)-based method and a PCR method using primers specific for a portion of the rfbE gene of E. coli O157. IMS is laborious and time consuming but ends up with the isolation of the pathogen. PCR is fast and less laborious, but it can only be used for screening purposes, so a further culture step is required to isolate the organism. For both fecal and meat samples, the IMS method was found to be more sensitive than the PCR. Furthermore, the detection efficiency of the PCR was influenced by the origin of the fecal sample and the type of meat. For sheep feces, the efficiency of the PCR appeared to be systematically lower than for cattle feces. And the efficiency of the PCR in detecting E. coli O157 in spiked samples of raw minced beef and dry-fermented sausages was systematically lower than in samples of filet americain. Based on this study, it can be concluded that both for animal feces and meat, IMS can be used more successfully to detect E. coli O157 than PCR, because IMS showed to be more sensitive and the outcome was not influenced by the type of animal feces or meat.     

Comparison of rapid enzyme-linked immunosorbent assay and immunomagnetic separation methods for detection of Escherichia coli O157 in fecal, hide, carcass, and ground beef samples

Rapid enzyme-linked immunosorbent assays (ELISAs) are approved for detection of Escherichia coil O157 in beef products. However, these kits have also been used in the industry to detect this pathogen on hides or in feces of cattle, although this use has not been validated. The objective of this study was to compare commercially available ELISAs (E. coli Now, Reveal, and VIP) with immunomagnetic separation along with selective media to detect E. coli O157 on hides, in feces, and in medium- and low-level-inoculated ground beef and carcasses (simulated by using briskets) samples. Naturally infected hide and fecal samples were subjected to both the immunomagnetic separation method and ELISAs for the detection of E. coli O157. Additionally, E. coli O157 inoculated and noninoculated ground beef and beef briskets were used to simulate meat and carcass samples. When comparing the detection results from the ELISAs (E. coli Now, Reveal, and VIP) to the immunomagnetic separation method, poor agreement was observed for fecal samples (kappa = 0.10, 0.02, and 0.03 for E. coli Now, Reveal, and VIP, respectively), and fair-to-moderate agreement was observed for hide samples (kappa = 0.30, 0.51, and 0.29 for E. coli Now, Reveal, and VIP, respectively). However, there was near-perfect agreement between the immunomagnetic separation method and ELISAs for ground beef (kappa = 1, 1, and 0.80 for E. coli Now, Reveal, and VIP, respectively) and brisket (kappa = 1, 1, and 1 for E. coli Now, Reveal, and VIP, respectively) samples. Assuming immunomagnetic separation is the best available method, these data suggest that the ELISAs are not useful in detecting E. coli O157 from hide or fecal samples. However, when ELISAs are used on ground beef and beef brisket samples they can be used with a high degree of confidence.     

高分辨率熔解曲线分析法检测食源性副溶血性弧菌

为建立一种快速鉴定副溶血性弧茵的HRM（高分辨率熔解曲线）real-timePCR法,以toxR为靶基因,结合特异性引物,通过优化反应体系及条件,进行特异性、敏感性及重复性评价,并初步应用于90份送检的鲜活海产品样本的检测。特异性试睑表明,该方法能选择性检测副溶血弧菌,Tm值为76．64±0．57℃;而与创伤弧菌、霍乱弧茵、金黄色葡萄球菌等多种海产品中常见的食源性病原菌没有交叉反应。灵敏度试验表明,该方法最少可检测toxR重组质粒的浓度为3．50×102copies／mL。重复性试验表明,同一样品于试验内及试验问的平均Tm值分别为76．53±0．35℃和76．74±0．52℃,变异系数分别为0．56±0．42％和1．11±0．73％。对90份鲜活海产品样本的检测证实该法可使阳性检出率从国标法的14．44%高至18．89％。本研究所建立的副溶血性弧菌HRMreal-ILrnePCR法具有特异性好、灵敏度高、重复性好的特点,能应用于食品样本的检测,具有很好的研究价值和应用前景。     

Real-time PCR assays for strain-specific quantification of probiotic strains in human faecal samples

Strain-specific real-time PCR assays were developed for the probiotic strains Bifidobacterium breve 99, Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii ssp. shermanii JS, used in combination, for quantification of the strains in human faecal samples. Unique randomly amplified polymorphic DNA (RAPD) sequences or a phage-related sequence of the strains were used as a basis for designing strain-specific primers and probes. The real-time PCR conditions were optimised and the specificities of the assays were confirmed by analysing DNA from related bacterial strains and from faecal samples originating from an intervention study. The assays allowed quantification with a linear range of six orders of magnitude, from 10¹ to 10⁷ target genomes per assay. The detection limit was 4 × 10⁵ genome copies per g of faeces. The strain-specific quantitative real-time PCR assays developed were suitable for identification and quantification of probiotic strains in human faecal samples in a controlled intervention trial.     

Comparison of culture, multiplex, and 5' nuclease polymerase chain reaction assays for the rapid detection of Yersinia enterocolitica in swine and pork products

Bacteriological culture was compared with multiplex and fluorogenic (TaqMan) polymerase chain reaction (PCR) assays for the detection of attachment invasion locus (ail)-bearing Yersinia enterocolitica in market weight swine, chitterlings, and ground pork. The TaqMan assay detected 1 pg of purified Y. enterocolitica DNA, whereas conventional gel-based PCR detected I ng of the same. The presence of ail-bearing Y. enterocolitica was tested in pork and feces artificially inoculated with Y. enterocolitica strain NADC 5561. The sensitivity limits of culture, multiplex, and TaqMan PCR assays were 4 x 10(3), 4 x 10(2), and 0.4 CFU/g, respectively, for the artificially inoculated pork. The sensitivity limits were 4 x 10(2), 4 x 10(2), and 0.4 CFU/g, respectively, for feces after a 48-h enrichment in a Yersinia selective broth. By the culture method, Y. enterocolitica was not detected in any of the swine specimens (n = 2,403) examined. By contrast, it was detected in 48 (2%) of the swine samples screened using the multiplex PCR and in 656 (27.2%) of these samples using the TaqMan assay. Using the culture method, Y. enterocolitica was detected in 8% of chitterling samples (n = 350) and in none of the ground pork samples (n = 350). It was identified in 27% of the chitterling samples using multiplex PCR and in 79% of these samples using the TaqMan assay. Ten percent of the ground pork samples contained Y. enterocolitica, as determined by the multiplex PCR, and 38% based on the TaqMan assay. The results suggest that pork products harbor more ail-bearing Y. enterocolitica than selected organs of freshly slaughtered hogs and that the TaqMan assay is more sensitive than either the multiplex PCR or traditional culture methods.     

Development of rapid real-time PCR and most-probable-number real-time PCR assays to quantify enterotoxigenic strains of the species in the Bacillus cereus group

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (10(2) to 10(7) CFU/g for cooked rice and chicken, 10(3) to 10(7) CFU/ml for milk, and 10(4) to 10(7) CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 10(0) CFU/ml. Both assays were tested with real food samples and shown to beconsiderably appropriate for B. cereus group detection and quantification.     

绿色魏斯氏菌实时荧光定量聚合酶链式反应检测方法的建立

以rpoA基因为靶基因,建立绿色魏斯氏菌SYBR Green Ⅰ实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）快速检测方法。针对rpoA基因设计特异性引物,建立绿色魏斯氏菌实时荧光定量PCR检测体系,通过特异性、灵敏度和重复性实验评价体系的检测效果,同时与常规PCR方法进行比较。结果表明：实时荧光定量PCR方法能够特异性检出绿色魏斯氏菌,对基因组DNA的检测灵敏度达到2.667×10－3 pg/μL,对纯培养物和模拟污染牛肉样品直接检测的灵敏度分别为30 CFU/mL和0.8 CFU/g;与常规PCR相比,实时荧光定量PCR检测的灵敏度是其1 000 倍;不同浓度样品独立重复实验循环阈值的标准差均小于1,变异系数在0.02%～1.28%之间。本研究所建立的绿色魏斯氏菌实时荧光定量PCR检测方法具有特异性好、灵敏度高、重复性好的特点,能够进行准确的定量检测,是快速检测绿色魏斯氏菌的有效手段。     

Comparison of the Diatheva STEC FLUO with BAX System Kits for Detection of O157:H7 and Non-O157 Shiga Toxin-Producing <Emphasis Type="Italic">Escherichia coli</Emphasis> (STEC) in Ground Beef and Bean Sprout Samples Using Different Enrichment Protocols

The aim of this study was to assess the performance of the Diatheva STEC FLUO and BAX System real-time PCR assays for detection of Shiga toxin-producing Escherichia coli (STEC) (stx1/stx2 and eae target genes) and O-group identification in ground beef and bean sprout samples. Ground beef (325 g or 25 g) and mung bean sprout (25 g) samples were inoculated with ~?10 CFU of the “top five” STEC (O157:H7, O26, O103, O111, and O145 as specified in EU regulation ISO13136:2012), enriched using different broths and incubation temperatures, and tested using the Diatheva and BAX real-time PCR assays. In ground beef, both molecular methods were able to detect the “top five” STEC, and lower Ct values were observed for the Diatheva kits compared to BAX System. The O111-contaminated samples gave negative results with both methods using mTSB?+?novobiocin for enrichment. In bean sprouts, both methods provided positive results, although detection was not possible using mTSB?+?acriflavin/cefsulodin/vancomycin for enrichment. In conclusion, the Diatheva and BAX methods detected the “top five” STEC in ground beef and bean sprouts when inoculated at low levels. Both assays provided equivalent results in terms of performance and reliability. Thus, the Diatheva kits are comparable to reference STEC-detection methods and could be used by the food industry to reliably detect the “top five” STEC.     

Distribution of human polyomaviruses, adenoviruses, and hepatitis E virus in the environment and in a drinking-water treatment plant

Large numbers of viruses are excreted in human feces and urine, which even at low concentrations may cause illness when ingested. Some of these viruses have not been traditionally monitored in terms of waterborne diseases and are considered emergent viruses, such as hepatitis E virus (HEV) and JC and BK polyomavirus (JCPyV and BKPyV). The high prevalence of human adenoviruses (HAdV) and polyomaviruses, which both show DNA genomes, in sewage from widely divergent areas has suggested the relevance of evaluating these viruses as possible indicators of viral contamination. The concentration of these viruses was analyzed in sewage and river water and after treatment in a drinking-water treatment plant including chlorination, flocculation, ozonation, and granulate active carbon (GAC) filtration. Samples of GAC-filtered water were collected before a second chlorination treatment. The river used as a source of fresh water presented an average concentration of 2.6 x 10(1) JCPyV and 4 x 10(2) HAdV GC (genome copies)/L. A removal of 2 logarithms (99%) of HAdV and JCPyV was observed in the drinking-water treatment plant. All the GAC-filtered water samples studied contained HAdV, with a mean value of 4.3 HAdV GC/L. HEV strains belonging to genotype 3 were frequently detected in low concentrations in urban sewage and in biosolids or sewage containing swine feces but not in the river water samples studied. The detection of viruses by molecular techniques is useful for genetically describe emergent viruses in community wastewaters and water supplies. Quantification of JCPyV and HAdV using quantitative real-time PCR (QPCR) may be useful for evaluating virus removal efficiency in water treatment plants and as an index of the virological quality of water and of the potential presence of human viruses.     

Rapid and reliable detection of Shiga toxin-producing Escherichia coli by real-time multiplex PCR

Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant Shiga toxin-producing E. coli (STEC) serogroups implicated in outbreaks of human foodborne illness worldwide. The increasing prevalence of these pathogens has important public health implications. Beef products have been considered a main source of foodborne human STEC infections. Robust and sensitive methods for the detection and characterization of these pathogens are needed to determine prevalence and incidence of STEC in beef processing facilities and to improve food safety interventions aimed at eliminating STEC from the food supply. This study was conducted to develop Taqman real-time multiplex PCR assays for the screening and rapid detection of the predominant STEC serogroups associated with human illness. Three serogroup-specific assays targeted the O-antigen gene clusters of E. coli O26 (wzy), O103 (wzx), and O145 (wzx) in assay 1, O45 (wzy), O111 (manC), and O121 (wzx) in assay 2, and O157 (rfbE) in assay 3. The uidA gene also was included in the serogroup-specific assays as an E. coli internal amplification control. A fourth assay was developed to target selected virulence genes for Shiga toxin (stx(1) and stx(2)), intimin (eae), and enterohemolysin (ehxA). The specificity of the serogroup and virulence gene assays was assessed by testing 100 and 62 E. coli strains and non-E. coli control strains, respectively. The assays correctly detected the genes in all strains examined, and no cross-reactions were observed, representing 100 % specificity. The detection limits of the assays were 10(3) or 10(4) CFU/ml for pure cultures and artificially contaminated fecal samples, and after a 6-h enrichment period, the detection limit of the assays was 10(0) CFU/ml. These results indicate that the four real-time multiplex PCR assays are robust and effective for the rapid and reliable detection of the seven predominant STEC serogroups of major public health concern and the detection of their virulence genes.     

Short communication: Effects of drying and analytical methods on nitrogen concentrations of feeds,feces, milk,and urine of dairy cows

Nitrogen concentrations in feeds, feces, milk, and urine samples were measured using 2 analytical methods following different drying procedures. Ten samples of corn silage, alfalfa silage, and concentrates collected from 2017 to 2018 at Krauss Dairy Research Center, The Ohio State University (Wooster), were used. A 4-d total collection digestion trial provided fecal samples from 10 cows (1 sample/cow), and another 10 cows were used to collect milk samples (1 sample/cow) and spot urine samples (1 sample/cow). Spot urine samples were acidified immediately to pH <3.0 when collected. Feed samples were oven dried (55°C) or lyophilized and analyzed using the Kjeldahl (KJ; copper sulfate as a catalyst) method and a combustion method (elemental analyzer; EA). Feces, urine, and milk samples were analyzed for N using the following methods: (1) fresh samples by KJ (referred to as wet KJ), (2) lyophilization (urine and milk for 8 h; feces for 120 h) followed by EA (LYO-EA), and (3) oven drying (milk and urine for 1 h; feces for 72 h at 55°C) followed by EA (OD-EA). Additionally, changes in N content of acidified urine at −20° over 180 d of storage were examined. Nitrogen concentrations in corn silage, alfalfa silage, and concentrates were greater for EA by 6.1, 4.8, and 8.3%, respectively, compared with KJ. Analysis of dried samples via EA compared with wet KJ resulted in lower fecal N content (27.8 vs. 29.3 g/kg of DM). Nitrogen concentration in fecal samples via KJ after lyophilization was lower by 5% compared with wet KJ but did not differ from LYO-EA, suggesting that N losses occurred during drying. Nitrogen determination with EA after drying of samples resulted in greater milk N (5.70 vs. 5.50 g/kg) and urinary N (9.16 vs. 9.06 g/kg) content compared with wet KJ. However, drying method (i.e., lyophilization vs. oven drying) did not affect N content of milk, urine, or feces. The use of EA resulted in lower percentage deviation of N content from duplicate sample assays for most samples (no difference was found for concentrate and fecal N), suggesting that EA was more precise than KJ. In conclusion, drying of feces caused N losses regardless of drying methods. For urine and milk samples, if drying is necessary (i.e., EA), oven drying at 55°C can be used rather than lyophilization. The N content was greater in feeds, milk, and urine when determined with EA versus KJ. In addition, N content in acidified and undiluted urine at −20° changed and should be analyzed within 90 d of storage. The results in the current study, however, did not account for laboratory-to-laboratory variation.     

Quantitative analysis of mitochondrial DNAs in macaque embryos reprogrammed by rabbit oocytes

In cloned animals where somatic cell nuclei and oocytes are from the same or closely related species, the mitochondrial DNA (mtDNA) of the oocyte is dominantly inherited. However, in nuclear transfer (NT) embryos where nuclear donor and oocyte are from two distantly related species, the distribution of the mtDNA species is not known. Here we determined the levels of macaque and rabbit mtDNAs in macaque embryos reprogrammed by rabbit oocytes. Quantification using a real-time PCR method showed that both macaque and rabbit mtDNAs coexist in NT embryos at all preimplantation stages, with maternal mtDNA being dominant. Single NT embryos at the 1-cell stage immediately after fusion contained 2.6 x 10(4) copies of macaque mtDNA and 1.3 x 10(6) copies of rabbit mtDNA. Copy numbers of both mtDNA species did not change significantly from the 1-cell to the morula stages. In the single blastocyst, however, the number of rabbit mtDNA increased dramatically while macaque mtDNA decreased. The ratio of nuclear donor mtDNA to oocyte mtDNA dropped sharply from 2% at the 1-cell stage to 0.011% at the blastocyst stage. These results suggest that maternal mtDNA replicates after the morula stage.     

Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA

Lactobacillus casei strain Shirota (LcS) has been used in the production of fermented milk products for many years and is one of the most intensively studied probiotics. To evaluate the ability of LcS to proliferate in human intestines after it has been ingested, we developed a PCR-based method to identify and quantify LcS using an LcS-specific primer set (pLcS) derived from a randomly amplified polymorphic DNA (RAPD) analysis. We confirmed the high specificity of the pLcS primer set in 167 bacterial strains (57 strains of L. casei and 110 other strains of bacteria commonly isolated from human feces). The method's ability to identify LcS matched that of an ELISA using a monoclonal antibody and a RAPD analysis in a representative sample of colonies cultured from human feces. The detection limit of quantitative PCR (qPCR) using pLcS was 10(4.6) per gram of feces. The number of LcS in feces detected with qPCR was highly and significantly correlated with the number of LcS added to fecal samples within the range of 10(4.6) to 10(9.6) per gram feces (r(2)=0.999, P<0.001). After 14 healthy subjects ingested 10(11.0) CFU of LcS daily for 7 days, 10(9.1+/-0.5) LcS g(-1) (mean+/-S.D.) was detected in the fecal samples of all subjects by qPCR, and 10(8.0+/-0.9) CFU g(-1) was detected by culture; these values were significantly different (P<0.001, paired t-test). After the subjects stopped ingesting LcS, fecal LcS counts obtained with both methods decreased daily. The values produced by the 2 methods might have differed because of an overestimation in the PCR analysis due to the presence of dead LcS cells or an underestimation in the culture system due to the use of selective culture media; however, dead LcS cells can also be beneficial as immunomodulators. We confirmed that qPCR with an LcS-specific primer set was a rapid and accurate method for determining the total amount of LcS in feces including dead or less active cells which could not be detected by culture method.     

Rapid and simultaneous quantitation of Escherichia coli 0157:H7, Salmonella, and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 10(2) to 10(9) CFU/ml for E. coli O157:H7, 10(3) to 10(9) CFU/ml for Salmonella, and 10(1) to 10(8) CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 10(5) CFU/g for E. coli O157:H7, 10(3) CFU/g for Salmonella, and 10(4) CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 10(3) CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli 0157:H7, Salmonella, and Shigella in food.