Universal Polymerase Chain Reaction Research Articles

Culture-independent Characterization of the Microbiome of Healthy Pulp

IntroductionAdvances in culture-independent molecular biotechnologies have driven a greater appreciation for the function of mutualistic microorganisms in the maintenance of states of health in humans. The purpose of this study was to test the long-held hypothesis that healthy pulp lack bacteria. MethodsStrict inclusion criteria were used to identify 10 pristine teeth from 10 healthy patients that were scheduled to be electively extracted in compliance with an orthodontic treatment plan. Using a rigorous disinfection protocol to isolate the operating field, the pulp space was accessed, and pulp tissue was collected in vivo from each tooth using a barbed broach. Genomic DNA was extracted from each pulp sample and analyzed for the presence of bacterial DNA using universal 16S ribosomal RNA polymerase chain reaction primers and MiSeq sequencing (Illumina, San Diego, CA) of community amplicons. ResultsOne hundred percent (10/10) of the tested pulp tissues demonstrated the presence of bacterial DNA, with a mean of 343 operational taxonomic units per sample (range, 191–479). These were derived from 12 genera in which Ralstonia, Actinetobacter, and Staphylococcus were predominant (43%–78% of total community). None of the negative-field controls and none of the instruments used in the study tested positive for the presence of contaminating DNA. ConclusionsThis study presents evidence to support the conclusion that the pulp spaces of pristine healthy teeth contain detectable bacterial DNA.

DNA barcoding reveals seasonal shifts in diet and consumption of deep-sea fishes in wedge-tailed shearwaters.

The foraging ecology of pelagic seabirds is difficult to characterize because of their large foraging areas. In the face of this difficulty, DNA metabarcoding may be a useful approach to analyze diet compositions and foraging behaviors. Using this approach, we investigated the diet composition and its seasonal variation of a common seabird species on the Ogasawara Islands, Japan: the wedge-tailed shearwater Ardenna pacifica. We collected fecal samples during the prebreeding (N = 73) and rearing (N = 96) periods. The diet composition of wedge-tailed shearwater was analyzed by Ion Torrent sequencing using two universal polymerase chain reaction primers for the 12S and 16S mitochondrial DNA regions that targeted vertebrates and mollusks, respectively. The results of a BLAST search of obtained sequences detected 31 and 1 vertebrate and mollusk taxa, respectively. The results of the diet composition analysis showed that wedge-tailed shearwaters frequently consumed deep-sea fishes throughout the sampling season, indicating the importance of these fishes as a stable food resource. However, there was a marked seasonal shift in diet, which may reflect seasonal changes in food resource availability and wedge-tailed shearwater foraging behavior. The collected data regarding the shearwater diet may be useful for in situ conservation efforts. Future research that combines DNA metabarcoding with other tools, such as data logging, may provide further insight into the foraging ecology of pelagic seabirds.

Optimal pcr primers for rapid and accurate detection of Aspergillus flavus isolates

Aspergillus flavus is among the most devastating opportunistic pathogens of several food crops including rice, due to its high production of carcinogenic aflatoxins. The presence of these organisms in economically important rice strip farming is a serious food safety concern. Several polymerase chain reaction (PCR) primers have been designed to detect this species; however, a comparative assessment of their accuracy has not been conducted. This study aims to identify the optimal diagnostic PCR primers for the identification of A. flavus, among widely available primers. We isolated 122 A. flavus native isolates from randomly collected rice strips (N = 300). We identified 109 isolates to the genus level using universal fungal PCR primer pairs. Nine pairs of primers were examined for their PCR diagnostic specificity on the 109 isolates. FLA PCR was found to be the optimal PCR primer pair for specific identification of the native isolates, over aflP(1), aflM, aflA, aflD, aflP(3), aflP(2), and aflR. The PEP primer pair was found to be the most unsuitable for A. flavus identification. In conclusion, the present study indicates the powerful specificity of the FLA PCR primer over other commonly available diagnostic primers for accurate, rapid, and large-scale identification of A. flavus native isolates. This study provides the first simple, practical comparative guide to PCR-based screening of A. flavus infection in rice strips.

Resolving Rapid Radiations within Angiosperm Families Using Anchored Phylogenomics.

Despite the promise that molecular data would provide a seemingly unlimited source of independent characters, many plant phylogenetic studies are still based on only two regions, the plastid genome and nuclear ribosomal DNA (nrDNA). Their popularity can be explained by high-copy numbers and universal polymerase chain reaction (PCR) primers that make their sequences easily amplified and converted into parallel datasets. Unfortunately, their utility is limited by linked loci and limited characters resulting in low confidence in the accuracy of phylogenetic estimates, especially when rapid radiations occur. In another contribution on anchored phylogenomics in angiosperms, we presented flowering plant-specific anchored enrichment probes for hundreds of conserved nuclear genes and demonstrated their use at the level of all angiosperms. In this contribution, we focus on a common problem in phylogenetic reconstructions below the family level: Weak or unresolved backbone due to rapid radiations ($\leqslant $10 million years) followed by long divergence, using the Cariceae-Dulichieae-Scirpeae (CDS, Cyperaceae) clade as a test case. By comparing our nuclear matrix of 461 genes to a typical Sanger-sequence dataset consisting of a few plastid genes (matK, ndhF) and an nrDNA marker (ETS), we demonstrate that our nuclear data is fully compatible with the Sanger dataset and resolves short backbone internodes with high support in both concatenated and coalescence-based analyses. In addition, we show that nuclear gene tree incongruence is inversely proportional to phylogenetic information content, indicating that incongruence is mostly due to gene tree estimation error. This suggests that large numbers of conserved nuclear loci could produce more accurate trees than sampling rapidly evolving regions prone to saturation and long-branch attraction. The robust phylogenetic estimates obtained here, and high congruence with previous morphological and molecular analyses, are strong evidence for a complete tribal revision of CDS clade. The anchored hybrid enrichment probes used in this study should be similarly effective in other flowering plant groups.

Clinical Utility of Universal PCR and its Real-world Impact on Patient Management

BackgroundOver the past decade, detection of bacterial and fungal DNA by universal polymerase chain reaction (PCR) has been increasingly used for organism identification in culture negative tissue samples. Few studies have assessed the diagnostic utility of this test in real-world clinical practice. The aim of this study was to assess the clinical performance of this test by examining available clinical information, test results and the impact on patient management.MethodsWe performed a single-center retrospective cohort study of patients who had clinical specimens sent for universal PCR from August 2013 to April 2016. Clinical data were extracted from medical records. Odds ratios were calculated and patients testing positive/negative were compared with univariate logistic regression. Sensitivity, specificity, positive predictive value and negative predictive values were calculated by comparing the test result with a gold standard composite final clinical diagnosis determined by 3 independent reviewers based on all available clinical information.Results71 tissue samples were included, of which 21(29.6%) were positive. 12 bacteria, 3 mycobacteria and 7 fungi were identified. The number of leukocytes in the gram stain (odds ratio, OR 1.57, P = 0.04) and presence of inflammation on histopathological examination (OR 5.69, P = 0.02) were found to be significantly associated with a positive result. The sensitivity, specificity, positive predictive value, and negative predictive values were 56%, 95%, 91% and 70% respectively. Management was altered in 22 patients, 9 of whom had a positive and 13 had a negative result.ConclusionThese findings suggest that the universal PCR assay has significant clinical utility, but the yield of this test can be optimized by careful patient/specimen selection. Utility was highest in patients with microscopic evidence of inflammation by gram stain or histopathlogical examination. Specificity was high. The use of this complex, difficult to interpret, and expensive test should be limited to infectious disease physicians incorporating all available clinical information to optimize performance.Disclosures All authors: No reported disclosures.

Bacterial profiling of healthy bone marrow using polymerase chain reaction technique

Background: The bone marrow of healthy individuals is conventionally considered sterile like other body fluids, but recently the scientists found genetic material from the bacteria inside their stem cells. The findings raise the possibility that other infectious agents may also reside in the bone marrow. Objectives: The study was planned to look for the presence of all type of bacteria by amplifying 16S rDNA sequences using eubacterial universal primers. Materials and Methods: A total of 18 bone marrow samples of apparently healthy individuals were collected from patients admitted having closed bone fractures. The DNA was isolated and was subjected to nested polymerase chain reaction (PCR) using Universal eubacterial 16S rDNA primers. The samples positive by universal PCR was further checked for the presence of Salmonella Typhi, Salmonella Paratyphi A, and/or Mycobacterium tuberculosis (Mtb) if any using a second nested PCR reaction. Results: A total of 16 (89%) samples could yield the desired amplicon through universal PCR. The secondary PCR of 16 samples, the desired amplicons were detected 3 (18.8%) for Mtb, 4 (25%) for S. Typhi, and 1 (6.2%) for S. Paratyphi A. Conclusions: Even in asymptomatic cases, other infectious agents such as Mtb, Salmonella spp., and many other microorganisms may be present as commensal.

Potential of high-throughput sequencing for broad-range detection of pathogenic bacteria in spices and herbs

A broad-range culture-independent method was developed and evaluated regarding its sensitivity of detection of pathogenic bacteria in spices and herbs, with focus on paprika powder. The method involved DNA extraction using cetyltrimethylammonium bromide (CTAB), 16S rDNA amplification using universal bacterial polymerase chain reaction, and high-throughput sequencing on Illumina MiSeq platform. The sensitivity of the method was evaluated with series of model samples contaminated at different levels with Salmonella enterica and Escherichia coli (as representatives of Gram-negative bacteria) and Staphylococcus aureus (as a representative of Gram-positive bacteria). For spices (paprika, black pepper), the method had a screening-level sensitivity with limits of detection in the range of 104–105 CFU/g, and a semi-quantitative response. Low sensitivity (LOD ≥107 CFU/g) was observed with herbs (oregano, parsley). The developed method demonstrated a good potential for microbiological screening of spices, with a prospect of further improvement of sensitivity based on progress in high-throughput sequencing technology.

Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing

We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)–PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC–PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%.

Accuracy of universal polymerase chain reaction (PCR) for detection of bacterial meningitis among suspected patients

IntroductionCentral nervous system (CNS) infections are life-threatening diseases caused by viral, bacterial, parasitic and fungal microorganisms. The aim of this study was to determine the accuracy of universal polymerase chain reaction (PCR) for the detection of bacterial meningitis among patients who were referred to Koodakan Hospital in Bandar Abbas because they were suspected of having the disease.MethodsThis study was conducted in 2013 on the patients who were admitted to Bandar Abbas’ Koodakan Hospital because they were suspected of having meningitis. A questionnaire, including demographic data, was completed for each patient. Universal PCR, Cerebrospinal fluid (CSF) analysis, and gram staining and cultures were done for all the patients. The data were analyzed using SPSS software.ResultsAmong the 100 patients studied 59 (59%) were male and 41 (41%) were female. No patient in our study had a positive smear and culture for meningitis. Among the patients with negative smears and cultures six (6%) had positive universal PCR, and 94 (94%) had negative universal PCR. Based on these results, PCR had 95% specificity and 100% negative predictive value for the prediction of meningitis. In 30 patients (30%), the biochemical analysis of CSF were in favor of meningitis. Among the 30 patients, six patients (20%) had positive universal PCR and 24 patients (80%) had negative universal PCR.ConclusionBased on our results, the universal PCR test is useful in the diagnosis of bacterial meningitis in children. We recommend using it in combination with other tests, such as CSF analysis, for diagnosis of bacterial meningitis.

Donor-Derived Cell-Free DNA Is a Novel Universal Biomarker for Allograft Rejection in Solid Organ Transplantation

BackgroundIn solid organ transplantation, sensitive real-time biomarkers to assess the graft health are desirable to enable early intervention, for example, to avoid full-blown rejections. During rejection, high amounts of graft-derived cell-free DNA (GcfDNA) are shed into the blood stream. The quantification of this GcfDNA in allotransplantation is considered to fulfill this need, because it can be measured with great precision and at reasonable cost. Patients and MethodsPatients from 2 ongoing studies in kidney (KTx) and heart (HTx) transplantation were monitored blinded on a scheduled basis, by means of a published universal droplet digital polymerase chain reaction to quantify the GcfDNA. ResultsImmediately after engraftment, GcfDNA reaches high values (>5% of total cfDNA), with a rapid decrease to values of <0.5% within 1 week. Living-related KTx recipients show lower initial values, reflecting the absence of preservation injury. Episodes of rejection in KTx and HTx are accompanied by a significant increase of GcfDNA (>5-fold) above values in patients without complications, occurring earlier than clinical or biochemical hints to rejection. One case of rejection, which became clinically suspect after 1 year and was proven with biopsy, showed a significant 10-fold increase 3 months earlier. ConclusionsThe quantification of GcfDNA has the potential to detect rejection episodes at early stages, when other means of diagnosis are not effective. The method's noninvasiveness enables the monitoring recipients at intervals that are desired to catch rejections at early actionable stages to prevent full-blown rejection. This biomarker will be particularly valuable in regimens to minimize immunosuppression.

Differential amniotic fluid cytokine profile in women with chorioamnionitis with and without funisitis

Objectives: To evaluate whether the amniotic fluid (AF) cytokine profile in women with chorioamnionitis may differentiate between those with and without funisitis.Subjects and methods: Forty women at high risk of chorioamnionitis were studied. Gestational age at study was 26.94. Amniocentesis, universal and specific polymerase chain reaction, and microbiological cultures were performed. AF IL-1β, IL-2, IL-4, IL-6, IL 8, IL-10, IL-12, TNF-alpha, IFN-gamma, and MMP-8 were measured by multiplex assay. After delivery, the placenta and umbilical cord were studied histologically. Comparisons were made between three groups: controls, and chorioamnionitis with and without funisitis.Results: In 25 cases, the histological findings were normal (61.5%). The remaining 15 composed of 9 cases of chorioamnionitis alone (9/40; 23.1%) and 6 cases of chorioamnionitis plus funisitis (6/40; 15.4%). All AF cytokine levels were significantly higher in the cases with chorioamnionitis in comparison to controls, except for IFN-gamma. The comparisons between the three groups showed significant differences between chorioamnionitis alone and chorioamnionitis plus funisitis in IL-1β, IL-6, IL-10, IL-12, IL-8, and TNF-alpha, with the levels being higher when funisitis was present. Logistic regression found a powerful predictive model for funisitis including the following cytokinesIL-4, IL-10, IL-12, and IL-8.Conclusions: Measurements of AF interleukins 4, 10, 12, and 8 allow to identify cases with funisitisin women at high risk of chorioamnionitis.

Cost-effectiveness of Babesia microti antibody and nucleic acid blood donation screening using results from prospective investigational studies.

Babesia microti causes transfusion-transmitted babesiosis (TTB); currently, blood donor screening assays are unlicensed but used investigationally. We developed a decision tree model assessing the comparative- and cost-effectiveness of B. microti blood donation screening strategies in endemic areas compared to the status quo (question regarding a history of babesiosis), including testing by: (1) universal antibody (Ab), (2) universal polymerase chain reaction (PCR), (3) universal Ab/PCR, and (4) recipient risk-targeted Ab/PCR. The model predicted the number of TTB cases, complicated TTB cases, cases averted, and quality-adjusted life years (QALYs). Economic outcomes included each strategy's per-donation cost, waste (number of infection-free units incorrectly discarded), and waste index (number wasted units/number true positives). Sensitivity analyses examined uncertainty in transmission probabilities, prevalence rates, and other key model inputs. Universal PCR in four endemic states would prevent 24 to 31 TTB cases/100,000 units transfused (pht) at an incremental cost-effectiveness ratio (ICER) of $26,000 to $44,000/QALY (transmission probability dependent) and waste index of zero. Universal Ab/PCR would prevent 33 to 42 TTB cases pht at an ICER of $54,000 to $83,000/QALY and waste index of 0.05. The questionnaire is most wasteful (99.62 units wasted pht; 208.62 waste index), followed by the risk-targeted strategy (76.27 units wasted pht; 0.68 waste index). The model predicted zero cases of TTB or complicated TTB with universal Ab/PCR (versus [33, 42] and [13, 18] pht, respectively [no screening]). Results are highly sensitive to transmission probabilities. Universal PCR in endemic states is an effective blood donation screening strategy at a threshold of $50,000/QALY. Using a higher cost-effectiveness ratio, universal Ab/PCR is the most effective strategy.

An unidentified cluster of infection in the Peruvian Amazon region.

Bartonella bacilliformis is the etiological agent of Carrion's disease, which is a neglected disease linked to people in low-socioeconomic populations in Andean valleys. An outbreak of B. bacilliformis was reported in a rural area of the Peruvian Amazon region. The aim of this study was to characterize this outbreak using molecular techniques. Fifty-three blood samples from patients diagnosed with Carrion's disease were analyzed by molecular tools, using both a Bartonella-specific polymerase chain reaction (PCR) and an universal PCR, both based on 16S rRNA gene amplification. Additional water samples from the area were also analyzed. Unexpectedly, the samples were positive only when the universal PCR was used. Although environmental contamination cannot be ruled out, the results showed that Sphingomonas faeni was the possible causative agent of this outbreak, and that water was the most feasible infection source. Diagnosis by clinical criteria or microscopy may lead to misdiagnosis. There is a need to include molecular tools in the routine diagnosis of febrile syndromes, including Carrion's disease.

Bacterial Community Dynamics of Salted and Fermented Shrimp based on Denaturing Gradient Gel Electrophoresis

The Korean traditional seafood jeotgal is consumed directly or as an additive in other foods to improve flavor or fermentation efficiency. Saeujot, made from salted and fermented tiny shrimp (SFS; Acetes japonicus), is the best-selling jeotgal in Korea. In this study, we reveal the microbial diversity and dynamics in naturally fermented shrimp by denaturing gradient gel electrophoresis (DGGE). The population fingerprints of the predominant microbiota and its succession were generated by DGGE analysis of universal V3 16S rDNA polymerase chain reaction (PCR) amplicons. Overall, 17 strains were identified from sequencing of 30 DGGE bands. The DGGE profiles showed diverse bacterial populations in the sample, throughout the fermentation of SFS. Staphylococcus equorum, Halanaerobium saccharolyticum, Salimicrobium luteum, and Halomonas jeotgali were the dominant bacteria, and their levels steadily increased during the fermentation process. Certain other bacteria, such as Psychrobacter jeotgali and Halomonas alimentaria appeared during the early-fermentation process, while Alkalibacterium putridalgicola, Tetragenococcus muriaticus, and Salinicoccus jeotgali appeared during the late-fermentation process. The members of the order Bacillales were found to be predominant during the fermentation of SFS. Furthermore, S. equorum was identified as the dominant bacterial isolate by the traditional method of culturing under aerobic and facultative anaerobic conditions. We expect that this information will facilitate the design of autochthonous starter cultures for the production of SFS with desired characteristic sensory profiles and shorter ripening times.

Heart Valve Culture and Sequencing to Identify the Infective Endocarditis Pathogen in Surgically Treated Patients

Testing excised valves in surgically treated infective endocarditis (IE) patients provides an opportunity to identify the microbial etiology of IE. Microbial sequencing (universal bacterial, mycobacterial, or fungal polymerase chain reaction followed by DNA sequencing) of valves can identify microorganisms accurately, but the value it adds beyond information provided by blood and valve cultures has not been adequately explored. Three hundred fifty-six patients who underwent surgery for active IE from January 1, 2010, to January 1, 2013, were identified from our cardiovascular information registry and outpatient parenteral antibiotic therapy registry. Their records were reviewed to identify 174 patients whose valves were sent for sequencing. The microbial etiology of IE was defined using comprehensive clinical, pathologic, and microbiological criteria. Blood culture, valve culture, and valve sequencing were examined to determine how frequently they identified the definitive cause of IE. Of the 174 patients, 162 (93%) had acute inflammation on histopathologic examination of their valves. Valve sequencing was significantly more sensitive than valve culture in identifying the causative pathogen (90% versus 31%, p < 0.001), and yielded fewer false positive results (3% versus 33%, p <0.001). The pathogen would not have been identified in 25 patients (15%) had it not been for valve sequencing. All the value provided by sequencing was attributable to bacterial DNA sequencing; mycobacterial and fungal sequencing provided no additional information beyond that provided by blood culture, histopathology, and valve culture. Valve sequencing, not valve culture, should be considered the primary test for identifying bacteria in excised cardiac valves.

Detection of Bacteria Bearing Resistant Biofilm Forms, by Using the Universal and Specific PCR is Still Unhelpful in the Diagnosis of Periprosthetic Joint Infections.

Intraoperative conventional bacteriological cultures were compared with different polymerase chain reaction (PCR) methods in patients with total joint arthroplasties. The isolated bacteria were investigated for biofilm formation, and the biofilm forming strains, in their planktonic and biofilm forms, were further tested for their antimicrobial resistance against several clinically important antimicrobials. Forty four bone and joint samples were included and classified as infected or non-infected according to standard criteria for periprosthetic hip and knee infections. For the bacteriological diagnosis, conventional culture, two types of universal PCR and species specific PCR for three selected pathogens (Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa) were applied. Biofilm formation determination was performed by the tissue culture plate method. Antimicrobial susceptibility of the planktonic bacteria was performed by the minimal inhibitory concentration determination and, of the biofilm forms, by the minimal inhibitory concentration for bacterial regrowth from the biofilm. Twenty samples were culture positive, with S. epidermidis, S. aureus, or P. aeruginosa. All PCR methods were very ineffective in detecting only one pathogen. All isolates were biofilm positive and their biofilm forms, were highly resistant. In this study, compared to PCR, culture remains the “gold standard.” The biofilm formation by the causative bacteria and the concomitant manifold increased antimicrobial resistance may explain the clinical failure of treatment in some cases and should be considered in the future for therapeutic planning.

Mobilization with granulocyte colony-stimulating factor blocks medullar erythropoiesis by depleting F4/80+VCAM1+CD169+ER-HR3+Ly6G+ erythroid island macrophages in the mouse

Similarly to other tissues, the bone marrow contains subsets of resident tissue macrophages, which are essential to maintain bone formation, functional hematopoietic stem cell (HSC) niches, and erythropoiesis. Pharmacologic doses of granulocyte colony-stimulating factor (G-CSF) mobilize HSC in part by interfering with the HSC niche-supportive function of BM resident macrophages. Because bone marrow macrophages are key to both maintenance of HSC within their niche and erythropoiesis, we investigated the effect of mobilizing doses of G-CSF on erythropoiesis in mice. We now report that G-CSF blocks medullar erythropoiesis by depleting the erythroid island macrophages we identified as co-expressing F4/80, vascular cell adhesion molecule-1, CD169, Ly-6G, and the ER-HR3 erythroid island macrophage antigen. Both broad macrophage depletion, achieved by injecting clodronate-loaded liposomes, and selective depletion of CD169(+) macrophages, also concomitantly depleted F4/80(+)VCAM-1(+)CD169(+)ER-HR3(+)Ly-6G(+) erythroid island macrophages and blocked erythropoiesis. This more precise phenotypic definition of erythroid island macrophages will enable studies on their biology and function in normal settings and on diseases associated with anemia. Finally, this study further illustrates that macrophages are a potent relay of innate immunity and inflammation on bone, hematopoietic, and erythropoietic maintenance. Agents that affect these macrophages, such as G-CSF, are likely to affect these three processes concomitantly.

Un‐nesting DNA Russian dolls – the potential for constructing food webs using residual DNA in empty aphid mummies

Constructing food-web assemblages comprising parasitoid wasps involves large field collections of hosts followed by labour-intensive rearing of the insects to evaluate the rates of parasitism along with morphological or molecular identification of the parasitoid species. This article presents research towards a new molecular method for the practical and accurate construction of aphid-based food webs. We hypothesize that parasitoid and hyperparasitoid DNA left inside aphid mummies after emergence of these third and fourth trophic-level guilds can be simultaneously detected using universal polymerase chain reaction (PCR) primers for nonspecific DNA amplification in combination with single-stranded conformation polymorphism (SSCP) analysis. Such a protocol theoretically allows food-web construction to be performed with no a priori knowledge of the species present. Moreover, the use of empty mummies circumvents rearing and minimizes labour and time in the field and laboratory. To test our hypothesis, we conducted DNA analyses on laboratory-produced parasitized aphids (mummies) from Myzus persicae and Brevicoryne brassicae (two important aphid pest species) after exposure to the parasitoid Diaeretiella rapae and the hyperparasitoid Asaphes vulgaris. DNA is amplifiable in empty aphid mummies for as long as 3weeks after parasitoid emergence. However, the simultaneous identification of several species in a single mummy sample was rare, which hinders the accurate inference of trophic links. DNA quality and relative quantity, together with preferential amplification, are potential explanations of current results. Technical refinements are needed to ensure full reliability and detection of complex trophic links. The use of PCR-SSCP for food-web construction is novel, and its potential to include an important number of different species is yet to be fully explored.

Evaluation of the Time Period for which Real-Time Polymerase Chain Reaction Detects Dead Bacteria

Real-time polymerase chain reaction (PCR) is currently widely used for the diagnosis of infections. We evaluated the time after treatment during which real-time PCR can detect dead bacteria. The presence of bacterial DNA was identified by real-time PCR through methicillin-resistant Staphylococcus (MRS)-PCR and universal PCR. Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, and Escherichia coli were each killed with alcohol, antibiotics, or heat treatment in vitro. The detection periods of MRS-PCR for MRSA treated by alcohol, vancomycin, linezolid, and heat were found to be less than 16, 8, 12, and 8 weeks, respectively. The detection period of universal PCR for S. epidermidis treated by alcohol, cefazolin, and heat was less than 20, 20, and 4 weeks, whereas that for E. coli was 8, 20, and 4 weeks, respectively. The presence of detectable bacterial DNA in infected arthroplasty patients before and after successful treatment was also assessed by MRS- and universal PCR. MRS-PCR was positive in 6 patients before treatment and all became negative after a mean interval of 20.8 weeks (95% confidential interval, 13.2 to 33.7) after treatment. Universal PCR detected remnant bacterial DNA in 4 patients at a mean of 15.2 weeks (95% CI, 12.4 to 18.0) after treatment and was negative in 7 patients at a mean of 17.3 weeks (95% CI, 10.6 to 24.0) after treatment. Our studies revealed that real-time PCR detects dead bacteria for several weeks, but this capability decreases with time and is likely lost by 20 weeks after treatment.

Molecular diversity of the equine caecal microbiota and its correlation to postprandial fermentation metabolites: A preliminary approach

In this work we address the short-term postprandial dynamic changes in the equine caecal microbial composition and the microbial fermentation metabolites in the caecum. Samples were collected every half hour for 8.5 hours postfeeding from two horses on two consecutive days. The caecal content of volatile fatty acids (VFAs), lactate, and ammonia (NH3N), as well as microbiota composition and pH were analyzed. Mixed Sangers sequences generated from universal polymerase chain reaction (PCR)-amplified 16S rRNA gene were used to analyze the microbiota composition. Three main phylogroups were identified and classified as Clostridium cluster XIVa, Clostridium cluster III, and lactic acid bacteria. Furthermore, we found that the composition of the caecal microbiota was closely associated with the VFA profile. Our main findings were that short-term microbial fluctuations occur in healthy hindgut fermenting horses as a response to feed intake.