rDNA PCR Research Articles

Comparison of ten different DNA extraction procedures with respect to their suitability for environmental samples

DNA extraction for molecular biological applications usually requires target optimized extraction procedures depending on the origin of the samples. For environmental samples, a range of different procedures has been developed. We compared the applicability and efficiency of ten selected DNA extraction methods published in recent literature using four different environmental samples namely: activated sludge from a domestic wastewater treatment plant, river sediment, anaerobic digestion sludge and nitrifying enrichment culture. We assessed the suitability of the extraction procedures based on both DNA yield and quality. DNA quantification was performed by both ultra violet (UV) spectrophotometry and fluorescence spectrophotometry after staining with PicoGreen.In our study, DNA yields based on UV measurement were overestimated in most cases while DNA yields from fluorescence measurements correlated well with the sample load on agarose gels of crude DNA. The quality of the DNA extracts was determined by gel electrophoresis of crude DNA and PCR products from 16S rDNA with the universal primer set 27f/1525r. It was observed that gel electrophoresis of crude DNA was not always suitable to evaluate DNA integrity and purity since interfering background substances (e.g. humic substances) were not visible. Therefore, we strongly recommend examining the DNA quality of both crude DNA and 16S rDNA PCR products by gel electrophoresis when a new extraction method is established.Summarizing, we found four out of ten extraction procedures being applicable to all tested samples without noticeable restrictions. The procedure G (according to the standard method 432_10401 of the Lower Saxony State Office for Consumer Protection and Food Safety) had the broadest application range over procedure J (published by Wilson, 2001). These were followed by procedures F (Singka et al., 2012) and A (Bourrain et al., 1999). All four extraction procedures delivered reliable and reproducible crude DNA and PCR products. From an economical point of view, all procedures tested during this study were cheaper compared to commercial DNA extraction kits.

Whipple’s endocarditis presenting as ulnar artery aneurysm; if you don’t look, you won’t find

A 54-year-old female patient presented to her local district general hospital with a painful, swollen left arm. Imaging revealed an ulnar artery aneurysm. The aetiology was embolic, with an echocardiogram...

Pleomorphic bacteria-like structures in human blood represent non-living membrane vesicles and protein particles

Although human blood is believed to be a sterile environment, recent studies suggest that pleomorphic bacteria exist in the blood of healthy humans. These studies have led to the development of “live-blood analysis,” a technique used by alternative medicine practitioners to diagnose various human conditions, including allergies, cancer, cardiovascular disease and septicemia. We show here that bacteria-like vesicles and refringent particles form in healthy human blood observed under dark-field microscopy. These structures gradually increase in number during incubation and show morphologies reminiscent of cells undergoing division. Based on lipid analysis and Western blotting, we show that the bacteria-like entities consist of membrane vesicles containing serum and exosome proteins, including albumin, fetuin-A, apolipoprotein-A1, alkaline phosphatase, TNFR1 and CD63. In contrast, the refringent particles represent protein aggregates that contain several blood proteins. 16S rDNA PCR analysis reveals the presence of bacterial DNA in incubated blood samples but also in negative controls, indicating that the amplified sequences represent contaminants. These results suggest that the bacteria-like vesicles and refringent particles observed in human blood represent non-living membrane vesicles and protein aggregates derived from blood. The phenomena observed during live-blood analysis are therefore consistent with time-dependent decay of cells and body fluids during incubation ex vivo.

Utility of a Direct 16S rDNA PCR and Sequencing for Etiological Diagnosis of Infective Endocarditis.

BackgroundCases of infective endocarditis (IE) require prompt etiological diagnosis for effective treatment. Molecular methods can aid in rapid and reliable diagnosis of culture-negative IE cases. We evaluated the utility of 16S rDNA PCR and sequencing in determining the causative agents of IE in valve tissues, especially when specimens were obtained after initiation of antimicrobial therapy.MethodsWe performed 16S rDNA PCR and sequencing in heart valve specimens and medical records review of 80 patients who underwent protocol-based cardiac surgery from 2013 to 2015. One patient did not meet the criteria for IE. Sixty-five (81.3%) and 14 pa-tients (17.5%) were diagnosed as having definite IE and possible IE, respectively. Blood and heart valve biopsy tissue were examined by using routine microbiological methods.ResultsBlood cultures in our hospital were IE-positive for 33 patients (41.8%), whereas 49 patients (62.0%) showed positive blood cultures when initial blood cultures performed at the referring hospital were included. Eighteen (22.8%) and 40 patients (50.6%) were IE-positive in valve tissue cultures and 16S rDNA PCR, respectively. Bacteria in the Streptococcus mitis group (n=26) were the most common etiological agents of IE. Eight (10.1%) culture-negative specimens tested positive by 16S rDNA PCR. In five of eight PCR-positive and culture-negative cases, fastidious or anaerobic organisms were the cause of IE.Conclusions Direct 16S rDNA PCR and sequencing can be used as a supplementary method to conventional blood and biopsy culture testing, especially in culture-negative IE cases that are negative for IE by culture.

Gut dysbiosis in mice genetically selected for low antibody production

BackgroundDysbiosis is linked to the cause of several human diseases, many of which having an immunity related component. This work investigated whether mice genetically selected for low or high antibody production display differences in intestinal bacterial communities, and consisted in the comparison of fecal 16SV6–V8 rDNA PCR amplicons resolved by temperature gradient gel electrophoresis (TGGE) of five each of low (LIII) and high (HIII) antibody producing mice. 16SV6 rDNA amplicons of 2 mice from each line were sequenced.ResultsLIII mice were grouped in a single TGGE cluster, displayed a low α-diversity, and were distinguished by low Firmicutes/Bacteroidetes ratio.ConclusionThe results suggest that genetically driven low antibody production in mice is associated with gut dysbiosis.

Endotoxemia is associated with acute coronary syndrome in patients with end stage kidney disease

BackgroundCardiovascular disease is the major cause of death in patients with end-staged kidney disease (ESRD). Most ESRD patients have systemic inflammation, and increasing the risk of cardiovascular event. Endotoxin derived from lipopolysaccharide of Gram negative bacteria accounts for 70% of intestinal bacteria, leading to release of proinflammatory cytokines and negative cardiovascular effect. Impaired intestinal barriers have been found in some ESRD patients, and may lead to bacteria translocation from gastrointestinal tract. We aim to investigate the association of endotoxemia in ESRD patients and acute coronary syndrome (ACS).MethodsWe collected serum from adult ESRD patients who presented to emergency department (ED) with ACS (30 patients) or without ACS (30 patients) as control from 11/01/2013 to 10/31/2014 in Chi Mei Medical Center in southern Taiwan. Clinical information and lab data were collected. We measured the endotoxin level of the serum of ESRD patients with or without ACS. We used real-time 16S rDNA PCR to detect possible bacteria in the blood of the patients.ResultsThe endotoxin level of ESRD patients with ACS (0.49 (±0.12) EU/mL) was significantly higher than that of ESRD patients without ACS (0.1 ± 0.08) (p < 0.01). However, the endotoxin level was not correlated with the troponin-I level (r = −0.12). Although endotoxin level was higher in ESRD patients with ACS, bacteria were not detected in the serum by using the real-time 16S rDNA PCR.ConclusionEndotoxin in ESRD patients with ACS was significantly higher than that without ACS. The result suggested that endotoxemia may have a contributory role to cardiovascular disease in ESRD patients.

Oomycota species associated with deciduous and coniferous seedlings in forest tree nurseries of Western Turkey

SummaryOccurrence and pathogenicity of Oomycota species causing root rot were investigated in 10 forest tree nurseries in western Turkey. Soil samples (129 in total) taken from the rhizosphere of symptomatic seedlings were baited for Oomycota using young leaves of Quercus suber, Rhododendron simsii and R. ponticum. Oomycota (178 isolates) were obtained by culturing on selective media, and identified using morphological methods followed by PCR and sequencing of the ITS rDNA and cox1 regions. Phytophthora aff. cactorum, P. citricola sensu lato, P. crassamura, P. syringae, Pythium aphanidermatum, Py. intermedium, Py. irregulare, Py. ultimum and Phytopythium vexans were common amongst the isolates. The highest diversity of Oomycota was found in the forest nurseries at Adapazarı‐Hendek and İzmir‐Torbalı. Pathogenicity tests showed that the isolates caused lesions on a range of host plants. The importance of these nursery infections in transferring potentially damaging oomycete species to Turkish forests is discussed.

Native valve endocarditis caused by Lactococcus garvieae: an emerging human pathogen

A 57-year-old man presented with native mitral valve endocarditis caused by Lactococcus garvieae, a known animal pathogen that is increasingly being reported as a cause of human infections. The organism...

Effects of Different Environmental Factors and Virulence Factors, dupA and iceA Genes, of Helicobacter pylori on Peptic Ulcer

Background: Helicobacter pylori is colonized in stomach and has a significant etiological role in different kinds of digestive diseases. Helicobacter pylori has the ability to devastate duodenal mucus causing peptic ulcer. Breakage of stomach lining is also seen more often among people who accustomed to consume tobacco, spicy (or sour) foods, alcohol, coffee, and tranquilizers as well as those who are exposed to heavy meals and stressful-life events (stress ulcer). Also, it seems dup A and ice A genes are involved in H. pylori etiology. Objectives: This study aimed to determine the relation of jhp0917 and jhp0918 genes, ABO blood type, and some environmental factors with peptic ulcer. Methods: In our study, 500 gastric mucosal biopsy samples from digestive tract were examined by rapid urease test (RUT) and 16s rDNA PCR test for H. pylori infection followed by histopathological examinations. The dup A and ice A genes were also evaluated by PCR. Results: In H. pylori colonized peptic ulcers, dup A gene showed no significant relation with peptic ulcer. On the other hand, iceA 1 (+)/ iceA 2 (-) genotype of H. pylori showed a strong association with peptic ulcer (P < 0.05). Conclusions: ABO blood group showed no association with H. pylori infection. Peptic ulcer had a significant relationship with consumption of tobacco (P < 0.05), consumption of tranquilizers (P < 0.05), and meteorism (P < 0.05).

What is broad-range 16S rDNA PCR?

PCRs have revolutionised the detection of bacteria in clinical samples since their widespread introduction in the 1990s.1 Quantitative PCR (qPCR), also known as specific PCR, involves the targeting of particular...

Organización y conservación de la colección de hongos patógenos y simbiontes de insectos y otros artrópodos del CEPAVE (CONICET-UNLP), La Plata, Argentina

The collection of fungal pathogens and symbionts of insects and other arthropods of the Centro de Estudios Parasitológicos y de Vectores, La Plata, Argentina, is unique because it preserves in vivo and in vitro cultures of fungal pathogens. This culture collection is open for research, teaching, consulting services, and strain deposit. It contains 421 strains belonging to 23 genera (16 Ascomycota, 4 Entomophthoromycotina, 2 Glomeromycota and 1 Oomycota), and the cultures are preserved by different methods such as cryopreservation in freezer at −20°C and −70°C, paper, distilled water and lyophilization. Fungi were isolated from insects, other arthropods, and soil (by using insect baits and selective media). Species were identified by morphological features and in a few strains by molecular taxonomy (PCR of rDNA). This collection is a reference center for species identification/certifications, research and teaching purposes, strain deposit, transference and consultancy services, and its overall goal is to preserve the fungal germplasm and ex situ diversity. Most of the strains are native of Argentina. The collection was originated in 1988 and is registered in the Latin American Federation for Culture Collections and in the World Federation of Culture Collections.

39 - Discitis in children: an evolution in diagnostic success with the use of 16S rDNA PCR

39 - Discitis in children: an evolution in diagnostic success with the use of 16S rDNA PCR

Comparison of culture-dependent and culture-independent molecular methods for characterization of vaginal microflora

To date, molecular methods that circumvent the limitations of traditional culture methods have not been used to describe the vaginal microflora in India. Here, we compared culture and culture-independent molecular methods in characterizing the vaginal microbiota in Indian women. Culture methods involved traditional cultivation on Rogosa and sheep blood agar, whereas culture-independent methods bypassed a culturing step by performing broadrange 16S rDNA PCR on DNA isolated directly from vaginal swabs. A total of 13 women were included in the study, of which five were characterized as healthy, two were bacterial vaginosis intermediate and six were bacterial vaginosis positive according to Nugent scoring. Lactobacillus jensenii was detected most frequently when using culture methods. On the other hand, Lactobacillus iners, which was not detected by culture methods, was the most common Lactobacillus sp. detected using cultivation-independent methods. We found little overlap between the species found using cultivation-dependent and cultivation-independent methods. Rather, culture-dependent and culture-independent methods were found to be complementary in describing the vaginal microflora among South Indian women. Culture-independent methods were found to be superior in detecting clinically relevant vaginal flora.

Optimization of the Isolation Procedure of Genomic DNA from a Mucus Laden Pulmonate Gastropod, Achatina fulica

Genomic DNA of good quality is a prerequisite in molecular biology studies involved in deciphering the information contained in the molecules that are chosen for studying the evolutionary interrelationships among taxa. Genomic DNA should be free of contaminants which may otherwise interfere while performing PCR. This is particularly essential while isolating genomic DNA from gastropods due to the presence of large amounts of mucopolysaccharides in their tissues. In the present study, we have developed a new method for obtaining high quality genomic DNA from a pulmonate snail Achatina fulica, which secretes a large amount of mucus. In this method, we have obtained good quality genomic DNA, compatible for downstream molecular biology applications like PCR and sequencing of 16S rDNA. The yield of the genomic DNA was 2790 µg/ml. This method may be extended for isolating high quality genomic DNA from different gastropods native to Indian sub-continent, which serves as a starting point for studying their phylogenetic affinities.

Histological Identification of Propionibacterium acnes in Nonpyogenic Degenerated Intervertebral Discs.

Purpose. Low-virulence anaerobic bacteria, especially the Propionibacterium acnes (P. acnes), have been thought to be a new pathogeny for a series of disc diseases. However, until now, there has been no histological evidence to confirm this link. The purpose of this study was to confirm the presence of P. acnes in nonpyogenic intervertebral discs via histological observation. Method. Degenerated intervertebral discs were harvested from 76 patients with low back pain and/or sciatica but without any symptoms of discitis or spondylodiscitis. The samples were cultured under anaerobic conditions and then examined using 16S rDNA PCR to screen for P. acnes. Samples found to be positive for P. acnes were stained with hematoxylin-eosin (HE) and modified Brown-Brenn staining and observed under a microscope. Results. Here, 16 intervertebral discs were found to be positive for P. acnes via 16S rDNA PCR and the prevalence was 21.05% (16/76). Among them, 7 samples had visible microbes stained with HE and modified Brown-Brenn staining. Morphological examination showed the bacteria to be Gram-positive and rod-shaped, so they were considered P. acnes. Conclusion. P. acnes is capable of colonizing some degenerated intervertebral discs without causing discitis, and its presence could be further confirmed by histological evidence. Targeting these bacteria may be a promising therapy method for some disc diseases.

Metagenomic Evaluation of Bacteria from Voles.

Voles (Arvicolinae, Rodentia) are known carriers of zoonotic bacteria such as Bartonella spp. and Francisella tularensis. However, apart from F. tularensis, the bacterial microbiome of voles has not previously been determined in Finland and rarely elsewhere. Therefore, we studied liver samples from 61 voles using 16S ribosomal RNA gene PCR analysis, followed by Sanger sequencing. Twenty-three of these samples were also studied with tag-encoded pyrosequencing. The samples originated from 21 field voles (Microtus agrestis), 37 tundra voles (Microtus oeconomus), and 3 bank voles (Myodes glareolus). With the more conventional 16S rDNA PCR analysis, 90 (33%) of the recovered 269 sequence types could be identified to genus level, including Bartonella, Francisella, Mycoplasma, Anaplasma, and Acinetobacter in 31, 15, 9, 9, and 9 sequences, respectively. Seventy-five (28%) matched best with sequences of uncultured bacteria, of which 40/75 could be classified to the order Clostridiales and, more specifically, to families Lachnospiraceae and Ruminococcaceae. Pyrosequencing from 23 samples revealed comparable and similar results: clinically relevant bacterial families such as Mycoplasmataceae, Bartonellaceae, Anaplasmataceae, and Francisellaceae were recognized. These analyses revealed significant bacterial diversity in vole livers, consisting of distinct and constant sequence patterns reflecting bacteria found in the intestinal gut, but including some known zoonotic pathogens as well. The molecular bacterial sequence types determined with the two different techniques shared major similarities and verified remarkable congruency between the methods.

Isolation and characterization of Lactobacillus paracasei LPC and phage ΦT25 from fermented milk

Infections by bacteriophages (phages) cause great economic losses in the dairy industry. Most dairy plants have encountered severe phage contamination, resulting in fermentation failure. This study was the first to isolate and characterize a lactic acid bacteria (LAB) phage from abnormal fermented milk in a Thailand factory. The results showed that only 1 LAB isolate was obtained. This LAB was identified by API 50 CHL, 16S rDNA sequence analysis and PCR using species-specific primers that were designed to differentiate the Lactobacillus casei group. From the above evidence, this isolate was identified as Lactobacillus paracasei. Small plaques were formed on double layer agar plates, and the Lb. paracasei phage was designated as ΦT25. Transmission electron micrographs showed that the phage ΦT25 had a hexagonal head (55 ± 4.6 nm in diameter) and a long noncontractile tail (184 ± 7.8 nm in length), indicating that it belongs to Bradley's group B or the Siphoviridae family. The one-step growth curve revealed a latent period of 55 min, with a burst size of approximately 38 phage particles per infected cell. Our host-range determination study demonstrated that this phage was incapable of cross-infecting with other genera or species of LAB tested. The genome size of the phage ΦT25 was estimated to be 41 kb. This phage did not survive heating to 72 and 90 °C and pH 2. The most effective chemical biocides were 75% ethanol, 800 ppm hypochlorite, 0.15%(v/v) peracetic acid, 1% (w/v) Virkon®, 5% Dettol® and 2-, 5% Decon® 90. This study has provided relevant information about the Lb. paracasei phage and recommendations for phage control in dairy plants.

Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria.

BackgroundRed blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria.ResultsFlow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents.ConclusionsThese studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators.

FIRST REPORT OF 'CANDIDATUS LIBERIBACTER SOLANACEARUM' IN COMMERCIAL CARROT SEEDS IN ITALY

‘Candidatus Liberibacter solanacearum’ (CaLsol) is an emerging bacterium, which is seed-borne in carrot (Bertolini et al., 2015). Exclusion of infected seeds is mandatory to prevent the introduction and the establishment of CaLsol in new areas, such as Italy. Here, we tested carrot seeds, sold in Italy during 2015, for CaLsol by analyzing three lots of ‘Berlicum’ (#1, #9 and #17) and one each of ‘Nantese’ (#6), ‘Flakkee’ (#11), ‘Bolero’ (#3) and ‘Mezza Lunga Nantese’ (#15). DNA was extracted using the QIAGEN DNeasy® Plant mini kit on 0.5 g of seeds ground in CTAB buffer. All samples were analyzed by real-time PCR of the 16S rDNA (Li et al., 2009) and conventional PCR of ISR16/23S (Ravindran et al., 2011) and 50S (Munyaneza et al., 2009) rDNAs. CaLsol DNA was detected, regardless of the method utilized, in all seed lots except ‘Bolero’. Sequence analysis identified two homogeneous groups of CaLsol isolates: I) lots #1, #9, #15 (GenBank accession Nos. KU041863 for ISR16/23S and KU041865 for 50SrDNA), and II) lots #6, #11, #17 (KT984852 for ISR16/23S and KU041864 for 50SrDNA). The single-nucleotide polymorphism analysis revealed that the first group was closely related to the CaLsol haplotype-E (except for having A at nucleotides 1620 and 1632 of ISR16/23S), while the second to the haplotype-D (except for having A at nucleotide 1648 of ISR16/23S, and T at nucleotides 920 and 1068 of 50SrDNA). The detection of CaLsol in different carrot seed cultivars commercialized in Italy highlights the requirement of harmonized and coordinated preventive measures to limit its spread.

A prospective multicenter evaluation of direct molecular detection of blood stream infection from a clinical perspective.

BackgroundRapid diagnosis and appropriate antimicrobial therapy are of major importance to decrease morbidity and mortality in patients with blood stream infections (BSI). Blood culture, the current gold standard for detecting bacteria in blood, requires at least 24–48 hours and has limited sensitivity if obtained during antibiotic treatment of the patient. The aim of this prospective multicenter study was to clinically evaluate the application of a commercial universal 16S/18S rDNA PCR, SepsiTest™ (PCR-ST), directly on whole blood.MethodsIn total 236 samples from 166 patients with suspected sepsis were included in the study. PCR-ST results were compared to blood culture, the current gold standard for detecting BSI. Because blood cultures can give false-negative results, we performed an additional analysis to interpret the likelihood of bloodstream infection by using an evaluation based on clinical diagnosis, other diagnostic tests and laboratory parameters.ResultsClinical interpretation of results defined the detected organism to be contaminants in 22 of 43 positive blood cultures (51.2 %) and 21 of 47 positive PCR-ST results (44.7 %). Excluding these contaminants resulted in an overall sensitivity and specificity of the PCR-ST of 66.7 and 94.4 % respectively. Of the 36 clinically relevant samples, 11 BSI were detected with both techniques, 15 BSI were detected with PCR-ST only and 10 with blood culture only. Therefore, in this study, SepsiTest™ detected an additional 71 % BSI compared to blood culture alone.ConclusionsMore clinically relevant BSI were diagnosed by molecular detection, which might influence patient treatment. An improved SepsiTest™ assay suited for routine use can have additional value to blood culture in diagnosing bacteremia in septic patients.