Polymerase Chain Reaction Mix Research Articles

First report of Leptosphaeria biglobosa 'brassicae' causing leaf spots in Eutrema japonicum in Yunnan, China.

Wasabi (Eutrema japonicum) is a root vegetable that is cultivated at large scales in southwestern China. In November 2021, approximately 40% of plants in a forested plantation in Dadishui, Yunnan Province, China (25.47°N, 103.22°E), showed leaf spot symptoms. The early symptoms were small black spots that gradually expanded into irregular brown to black lesions (0.5-1.5 cm), which were restricted by leaf veins. Yellow halos were observed at the outer edges of necrotic lesions. To identify the causal agent, we collected 20 diseased leaves and obtained fungal isolates from symptomatic leaf tissues. Following surface sterilization with 75% ethanol for 30 s, the tissues were cultured on potato dextrose agar (PDA) plates and incubated at 25°C under a 12 h light/12 h dark light cycle. After 7 days of incubation, a total of 12 isolates were obtained through single-spore culture. All isolates had similar colony morphology, and produced fluffy white mycelia and yellow pigment after 1 week of PDA culture at 25°C, and blackish- brown mycelium, tan pigment, and conidia after 2 weeks. The conidia were hyaline and cylindrical, with an average size of 4.6 μm × 2.2 μm. These morphological characteristics similar to the description of Leptosphaeria biglobosa (Shoemaker et. al, 2001) and Leptosphaeria maculans (Vincenot et al. 2008). Genomic DNA was extracted from mycelium of isolate SK-1, which was harvested from 10-day-old PDA culture using a FAST plant genomic DNA Extraction Kit (Biomed, China), following the manufacturer's instructions. The species-specific primers LbigF, LmacF, and LmacR (Liu et al. 2006) were used for identification via polymerase chain reaction (PCR). A 444-bp fragment characteristic of L. biglobosa 'brassicae' (Lbb), and a 330-bp of L. maculans 'brassicae' (Lmb) were amplified, respectively. Internal transcribed spacer (ITS) sequences (592 bp), part of the 5' end of beta-tubulin (968 bp), and actin (899 bp) were also amplified using the primers ITS1/ITS4, BT1/BT2, and ACTF/ACTR (Vincenot et al. 2008), respectively. PCR was performed in a volume of 25 μL containing 12.5 μL 2 × T5 Super PCR Mix (Tsingke Biotech, Beijing, China), 1 μL 10 μM primer (Tsingke Biotech), 1 μL DNA template, and an aliquot of sterile water to attain the total volume. The thermal cycler settings were 5 min at 98°C; 35 cycles of 10 s at 98°C, 10 s at 58°C, and 30 s at 72°C; and extension for 2 min at 72°C. The ITS sequence of isolate SK-1 (GenBank accession no. OQ216838), the partial β-tubulin gene sequence (OQ241183), and the actin gene sequence (OQ241184) indicated 100% query cover and 100% identity with L. biglobosa (DQ458906), Lbb strain B3.6 (AY748995), and Lbb strain 2379-4 (AY748949), respectively. Phylogenetic analysis (King et al. 2022) also identified of isolate SK-1 as Lbb. To determinate its pathogenicity, isolate SK-1 was grown on PDA incubated at 28°C for 2 weeks, and conidial suspensions were prepared at a concentration of 106 conidia/mL. Then, 15 leaves of 4-month-old E. japonicum seedlings were needle-wounded on the front and inoculated by syringe injection of 10 μL of the appropriate conidial suspension. We used 10 μL of the sterilized distilled water as the control under forest growth conditions. All inoculation sites were covered with cotton strips and moistened with 1.0 mL sterile water to maintain humidity. After 12 days of incubation, the leaves developed symptoms similar to those observed in the field, and the fungus was reisolated from diseased leaves, whereas the controls remained healthy. Based on these results, we identified L. biglobosa 'brassicae' as the causal agent of leaf spot on E. japonicum in China. This fungus has been reported to cause blackleg in many Brassica crops in China such as Brassica napus (Fitt et al. 2006), Brassica oleracea (Zhou et al. 2019), B. juncea var. tumida (Deng et al. 2020), Brassica rapa subsp. pekinensis (Yu et al. 2021). To the best of our knowledge, this is the first report of L. biglobosa causing leaf spots in E. japonicum in China. Our data provide a basis for disease management in E. japonicum production in China.

Prevalence and diagnosis of retroviral infections in cats

Cat retroviruses, feline immunodeficiency virus and feline leukemia virus cause diseases that have a wide range of clinical manifestations and forms of infection. Aretrospective analysis of data from the last three years in the studied hospitals showed that the infection of cats with retroviruses is 32.6% for FeLV and 13.6% for FIV from the number of infectious diseases. Such high rates indicate that one third of cats coming to veterinary clinics with infectious diseases are infected with retroviruses. These are primarily animals at risk that have free range. Early detection of such animals by a veterinarian is important for effective treatment and prevention of infection. We have adapted the algorithm for the diagnosis of retroviral infections in cats to domestic conditions and developed practical recommendations for veterinarians on the effective choice of laboratory methods for the diagnosis of retroviral infections. It was found that in clinics, the initial study of whole blood samples from infectiously infected cats was performed using commercial serological test systems VetExpert FIVAb + FeLVAg. After the results of the first stage of the study, the protocol of the nested polymerase chain reaction (PCR) was tested. DNA isolation was performed using the IndiSpin Pathogen Kit. OneTaq® 2X Master PCR mix with Standard Buffer and specific oligonucleotides were used in the study. Proviral DNA was detected in 100% of blood samples for the respective pathogens FIV and FeLV. All animals that were pre-diagnosed with FIV and VLC infection at the first stage of diagnosis were diagnosed with proviral DNA and confirmed at the second stage of diagnosis. The use of the nested PCR method significantly increases the sensitivity and specificity of the study. The first stage of diagnosis allows a veterinarian to determine the infectious status of the animal on the basis of which to choose a treatment strategy and prevent transmission of the pathogen. The second stage of diagnosis allows you to clarify the previous diagnosis and establish the form of infection. The presence of a clear and understandable diagnostic algorithm is necessary for effective control of FIV and FeLV. Key words: polymerase chain reaction, feline viral leukemia, feline immunodeficiency virus, molecular diagnostics, retroviral infections, diagnostic algorithm.

A 3D-Printed Microfluidic Device for qPCR Detection of Macrolide-Resistant Mutations of Mycoplasma pneumoniae.

Mycoplasma pneumonia (MP) is a common respiratory infection generally treated with macrolides, but resistance mutations against macrolides are often detected in mycoplasma pneumoniae in China. Rapid and accurate identification of mycoplasma pneumoniae and its mutant type is necessary for precise medication. This paper presents a 3D-printed microfluidic device to achieve this. By 3D printing, the stereoscopic structures such as microvalves, reservoirs, drainage tubes, and connectors were fabricated in one step. The device integrated commercial polymerase chain reaction (PCR) tubes as PCR chambers. The detection was a sample-to-answer procedure. First, the sample, a PCR mix, and mineral oil were respectively added to the reservoirs on the device. Next, the device automatically mixed the sample with the PCR mix and evenly dispensed the mixed solution and mineral oil into the PCR chambers, which were preloaded with the specified primers and probes. Subsequently, quantitative real-time PCR (qPCR) was carried out with the homemade instrument. Within 80 min, mycoplasma pneumoniae and its mutation type in the clinical samples were determined, which was verified by DNA sequencing. The easy-to-make and easy-to-use device provides a rapid and integrated detection approach for pathogens and antibiotic resistance mutations, which is urgently needed on the infection scene and in hospital emergency departments.

A CRISPR/Cas9 eraser strategy for contamination-free PCR end-point detection.

Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end‐point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse‐transcription PCR (RT‐PCR) system to achieve contamination‐free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre‐incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT‐PCR and PCR systems, showed high‐fidelity detection of SARS‐CoV‐2 and African swine fever virus with a convenient strip test.

Effects of PCR inhibitors on mRNA expression for human blood identification.

Detection of body fluid-specific mRNAs, such as those specific for blood, using real-time polymerase chain reaction (PCR) has become a useful tool in forensic science. Blood stains often contain PCR inhibitors that may be co-extracted with RNA and adversely affect PCR. The effects of inhibitors on the detection of mRNA markers for blood identification, namely, hemoglobin beta (HBB) and actin beta, were examined herein. Inhibitors were added to a real-time PCR mix, reverse transcription mix, and blood samples before RNA extraction, and the following parameters: Ct, delta Ct (dCt), and melting temperature (Tm) values, were monitored. Hematin, humic acid, indigo carmine, and tannic acid were used as PCR inhibitors. The results showed that Ct values for HBB in samples containing inhibitors in their real-time PCR mix increased in a concentration-dependent manner, and were undetectable at higher concentrations. Moreover, Ct values for HBB in tannic acid-spiked samples reached a maximum once, and inhibition decreased at higher concentrations. dCt values increased in hematin-spiked samples, but decreased in tannic acid-spiked samples. Tm values decreased following the addition of each inhibitor. The reverse transcription reaction was scarcely inhibited at concentrations that markedly affected real-time PCR. The complete removal of inhibitors added to blood was difficult. However, the observed inhibitory effects were weaker than those when inhibitors were added to the PCR cocktail. PCR inhibition was effectively reduced by repurification of complimentary DNA with DNA extraction kits. These results will assist examiners in deducing contaminating inhibitors and selecting an appropriate method to remove them.

Development of a dry-reagent mix-based polymerase chain reaction as a novel tool for the identification of Acinetobacter species and its comparison with conventional polymerase chain reaction.

BACKGROUND:Nosocomial infections are often caused by multidrug-resistant bacteria and the incidence is increasing. Acinetobacter, a Gram-negative bacillus, is commonly associated with the use of intravascular catheterization and airway intubation. Polymerase chain reaction (PCR) for identification of Acinetobacter baumannii from samples has been standardized that use conventional wet-reagent mix. We have designed and optimized a dry-reagent mix for identification of Acinetobacter species by PCR. The dry-reagent mix can be stored at room temperature, has less chances of contamination, and thus can be used at point-of-care diagnosis.AIM AND OBJECTIVE:The present work was focused on comparing the sensitivity and specificity of dry-reagent PCR mix over conventional wet-reagent PCR mix for identification of Acinetobacter species.MATERIALS AND METHODS:Conventional wet-reagent mix based and dry-reagent mix based PCR were carried out for the DNA isolated from Acinetobacter species. The latter was also applied directly on bacterial growth without prior DNA extraction process. Equal numbers of bacterial isolates other than Acinetobacter species were also subjected to identification by the same protocols for determining the sensitivity and specificity of the test.RESULTS:The Acinetobacter species showed amplification of the target rpoB gene and the band was observed at 397 bp. The dry-reagent PCR mix results matched completely with the conventional wet-reagent PCR mix assay. All the non-Acinetobacter isolates were negative for the PCR. This indicates that the test is highly specific. The dry-reagent mix also contained an enzyme resistant to PCR inhibitors and capable of amplifying DNA directly from cells.CONCLUSION:Performance of dry-reagent PCR mix without the need for DNA extraction and preparation of a PCR mix proved to be more sensitive and reduce the handling error, minimizes the time, manual work, and skilled labor.

Effect of surface functionalisation on the interaction of iron oxide nanoparticles with polymerase chain reaction

The combination of nanoparticles with the polymerase chain reaction (PCR) can have benefits such as easier sample handling or higher sensitivity, but also drawbacks such as loss of colloidal stability or inhibition of the PCR. The present work systematically investigates the interaction of magnetic iron oxide nanoparticles (MIONs) with the PCR in terms of colloidal stability and potential PCR inhibition due to interaction between the PCR components and the nanoparticle surface. Several types of MIONs with and without surface functionalisation by sodium citrate, dextran and 3-aminopropyl-triethoxysilane (APTES) were prepared and characterised by Transmission Electron Microscopy (TEM), dynamic light scattering (DLS) and Fourier Transform Infrared (FT-IR) spectroscopy. Colloidal stability in the presence of the PCR components was investigated both at room temperature and under PCR thermo-cycling. Dextran-stabilized MIONs show the best colloidal stability in the PCR mix at both room and elevated temperatures. Citrate- and APTES-stabilised as well as uncoated MIONs show a comparable PCR inhibition near the concentration 0.1mgml−1 while the inhibition of dextran stabilized MIONs became apparent near 0.5mgml−1. It was demonstrated that the PCR could be effectively carried out even in the presence of elevated concentration of MIONs up to 2mgml−1 by choosing the right coating approach and supplementing the reaction mix by critical components, Taq DNA polymerase and Mg2+ ions.

Advanced and conventional molecular techniques in the diagnosis of Mycoplasma pneumoniae in patients with respiratory tract infection

Background:-M. pneumoniae is an important human pathogen that produces community-acquired respiratory tract infection. Diagnosis of M. pneumoniae infection is challenging and crucial for the timely initiation of the effective antibiotic therapy.Objective: This study has been undertaken to detect M. pneumoniae in respiratory samples (throat swabs, throat wash and sputum) in patients with respiratory tract infection qualitatively by conventional polymerase chain reaction (PCR). Also, more advanced one, real time PCR was used to determine mycoplasmal target gene qualitatively and quantitatively.Patients and methods: The study was performed on Seventy-five patients and thirty healthy subject as control. Genomic DNA was extracted and M. pneumoniae target gene (lipoprotein gene) was amplified using conventional PCR. Negative, positive controls and internal controls were involved in each experimental run. The amplified products were analyzed in 2% agarose gel and visualized using Red safe staining. In real time PCR, specific primer and probe mix depending on TaqMan® principle was used to detect P1 adhesion gene through FAM channel. A fluorogenic probe was included in the same reaction mixture which consists of a DNA probe labeled with a 5`-dye and a 3`-quencher. During PCR amplification Data were analyzed using Smart-cycler software and M. pneumoniae DNA copy number was estimated from the cross point threshold relative to positive standard.Results: Thirty five patients(45.5%) were positive by PCR and Thirty two (42.6 %) were positive by Real-time PCR. The highest rate of infection by using tow molecular methods were of less than 20 years of age. The quantity of M. pneumoniae DNA target gene in positive Real-time PCR were ranged between 10-2000copies/μl.Conclusion: The study concluded that both of molecular techniques conventional and real-time PCR are a rapid, reliable and ideal in diagnosis of M. pneumoniae using throat swabs, throat wash and sputum samples.

Single cell analysis exposes intratumor heterogeneity and suggests that FLT3-ITD is a late event in leukemogenesis

FMS-like tyrosine kinase 3 receptor-internal tandem duplication (FLT3-ITD) commonly occurs in acute myeloid leukemia and is considered rare in acute lymphocytic leukemia. Acute leukemia has poor prognosis, mainly due to relapse. Standard FLT3-ITD diagnostic techniques are based on genomic polymerase chain reaction and have recently incorporated GeneScan (Applied Biosystems, Foster City, CA) to identify variations of the FLT3 gene. As this is an average-based assay utilized in a heterogeneous leukemic cell population, we hypothesized that cells of acute leukemia, considered FLT3-ITD-negative by standard methods, could possess a fraction of FLT3-ITD-positive cells. The present study employed single cell mutation analysis to evaluate the FLT3-ITD status in newly diagnosed acute myeloid leukemia (n = 5) and acute lymphocytic leukemia (n = 3) patients. A total of 541 single leukemic cells and 36 mononuclear cells from healthy volunteers were analyzed. Seven patients, considered FLT3-ITD-negative according to bulk DNA analysis, appeared to possess a small fraction of FLT3-ITD-positive cells based on single cell analysis. Moreover, this approach revealed the heterogeneity of the tumor as evident by different FLT3-ITD mutations present in the same patient. The presence of a minor clone carrying FLT3-ITD in almost all patients tested provides evidence that this lesion is a common late event in leukemogenesis. Additionally, 3 relapsed patients demonstrated loss of heterozygosity of the normal allele, affecting 25%-100% of the cells found to be FLT3-ITD-positive. Though further clinical testing is warranted, these findings may have implications on the prognostic significance of FLT3-ITD and the use of targeted therapy.

A novel semi-multiplex PCR assay for identification of tropical eels of genus Anguilla in Indonesian waters

A one-step semi-multiplex polymerase chain reaction (PCR) is proposed to distinguish seven species and subspecies of tropical eels, i.e., Anguilla celebesensis, A. interioris, A. nebulosa nebulosa, A. marmorata, A. borneensis, A. bicolor bicolor, and A. bicolor pacifica, known in Indonesian waters. Seven pairs of species-specific primers, including two forward and seven reverse sequences, were designed after alignment of published and new complete mitochondrial cytochrome b (1,140 bp) and 16S ribosomal RNA (rRNA) (1,120 bp) sequences. All species-specific primer pairs are included in one PCR mix, but only one pair of them can amplify a specific fragment from the template DNA that is analyzed. The semi-multiplex PCR amplified a fragment of 230 bp for A. b. bicolor, 372 bp for A. n. nebulosa, 450 bp for A. borneensis, 620 bp for A. marmorata, 670 bp for A. b. pacifica, 720 bp for A. celebesensis, and 795 bp for A. interioris, which are then separated by DNA agarose gel electrophoresis.

Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis

Transient receptor potential (TRP) channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca(2+)](i)) through TRPC2 and TRPC3. Because modulation of [Ca(2+)](i) is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild-type littermate controls. Although the erythropoietin-induced increase in [Ca(2+)](i) was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenylhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with a significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. Although erythropoietin-induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis, which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.

PCR, principles, advantages and pitfalls: focus in some viral infections of the anterior segment of the eye

AbstractGene amplification using polymerase chain reaction (PCR) has become the gold standard for microbiological diagnosis in eye diseases, particularly in those related to viruses. Even if the specificity and the sensibility indexes are much higher than conventional methods, gene amplification may present some limits. For example, uveitis with low grade viral replication may induce false‐negative PCR results while assessment of the immune charge in aqueous may be positive in such cases. For infections of the ocular surface, PCR is usually much more effective than isolation of the virus in cell culture, but the high sensibility may inversely induce false‐positive results. For example, continuous shedding of herpes simplex in tears is known to occur regularly even in asymptomatic patients, which could mask the real diagnosis of any other cause of ocular redness. Even latest technologies, such as real time quantitative PCR, are not completely satisfying since a misuse of this very sensitive technique may also lead to false positive results if effective controls are not systematically used for each assay, due to the self‐fluorescence of the PCR mix when the number of thermal cycles increases. Inversely, vital dies, anesthetics eye drops or non‐optimal tissues purification may lead to false negative results due to the inhibition of the polymerase reaction.

A Micro Polymerase Chain Reaction Module for Integrated and Portable DNA Analysis Systems

This work deals with the design, fabrication, and thermal characterization of a disposable miniaturized Polymerase Chain Reaction (PCR) module that will be integrated in a portable and fast DNA analysis system. It is composed of two independent parts: a silicon substrate with embedded heater and thermometers and a PDMS (PolyDiMethylSiloxane) chamber reactor as disposable element; the contact between the two parts is assured by a mechanical clamping obtained using a Plastic Leaded Chip Carrier (PLCC). This PLCC is also useful, avoid the PCR mix evaporation during the thermal cycles. Finite Element Analysis was used to evaluate the thermal requirements of the device. The thermal behaviour of the device was characterized revealing that the temperature can be controlled with a precision of ±0.5°C. Different concentrations of carbon nanopowder were mixed to the PDMS curing agent in order to increase the PDMS thermal conductivity and so the temperature control accuracy.

Direct Polymerase Chain Reaction-Based Detection of Cercospora beticola in Field Soils.

Cercospora beticola, the causal agent of Cercospora leaf spot of sugar beet, survives as pseudostromata in infected sugar beet residues in the soil. Under optimal conditions, overwintering propagules germinate and produce conidia that are dispersed as primary inoculum to initiate infection in sugar beet. We developed a polymerase chain reaction (PCR) technique for rapid detection of C. beticola in field soils. Total DNA was first isolated from soil amended with C. beticola culture using the PowerSoil DNA Kit. The purified DNA was subjected to PCR in Extract-N-Amp PCR mix with CBACTIN primers over 35 cycles. The amplified products were resolved and compared by electrophoresis in 1% agarose gels. The PCR fragment size of C. beticola from the amended field soil correlated in size with the amplicon from control C. beticola culture DNA extract. Additionally, sample soils were collected from sugar beet fields near Sidney, MT and Foxholm, ND. Total DNA was extracted from the samples and subjected to PCR and resolved as previously described. The amplicons were purified from the gels and subjected to BigDye Terminator Cycle sequencing. All sequences from field soils samples, C. beticola-amended field soil, and pure culture were compared by alignment with a C. beticola actin gene sequence from GenBank. The result of the alignment confirmed the amplicons as products from C. beticola. Rapid screening for the presence of C. beticola in the soil by PCR will improve research capabilities in biological control, disease forecasting, and management of this very important sugar beet pathogen.

Population genetic structure of the salmon louse,Lepeophtheirus salmonis(Krøyer) on wild and farmed salmonids around the Pacific coast of Canada

The population genetic structure of the salmon louse (Lepeophtheirus salmonis: Caligidae) is of considerable interest because of indirect evidence suggesting transmission of this ectoparasite between wild salmon and farmed salmon (reviewed by Boxaspen 2006). Consequently several studies have looked for farm-speci¢c molecular markers that would allow the path of transmission to and from wild ¢sh to be traced. Unfortunately, an early report of RAPD markers that were speci¢c to Scottish salmon farms (Todd,Walker,Woli, Northcott,Walker, Ritchie, Hoskins, Abbott & Hazon 1997) was not found in a second RAPD study (Dixon, Shinn & Sommerville 2004) or in a more extensive study using six microsatellite markers (Todd, Walker, Ritchie, Graves & Walker 2004). Nevertheless, interest in ¢nding farmspeci¢c molecular markers continues. It is still not possible to follow the salmon lice larvae during the 4^14-day period they spend in the plankton before they infect a host (Johnson & Albright 1991). We therefore tested the research hypothesis that population genetic structure existed among salmon lice populations collected from diierent geographical regions of British Columbia, Canada, because of restricted gene £ow. Samples of the salmon louse, L. salmonis, were obtained from three diierent wild Paci¢c salmon species by sports ¢shing and during creel (sports ¢shery catch) surveys and from farmed Atlantic salmon by netting (Table 1). Sea lice were removed from each salmon host with forceps and preserved in 95% ethanol. To test for population genetic structure with the largest sample size possible, we decided to use a fragment of the mitochondrial cytochrome c oxidase subunit 1 (COI) (Folmer, Black, Hoeh, Lutz & Vrijenhoek1994) that has been extensively used for DNA barcoding (Hebert, Cywinska, Ball & deWaard 2003). This allowed us to make use of a large number of existing salmon louse COI sequences and to easily compare the sequence divergence that we observed among diierent clades with that of other species of animals (Hebert et al. 2003). Template DNA was extracted from individual salmon lice using modi¢ed proteinase K methods. A 1mm piece of ethanol-preserved tissue was obtained fromeach specimenand placed directly into 96-well plates containing lysis buier and proteinase K. Subsequent DNA extraction used a glass ¢bre protocol (Ivanova, deWaard & Hebert 2006). A 710-bp fragment of the COI genewas polymerase chain reaction (PCR) ampli¢ed using the primer pairs LCO1490 and HCO2198 (Folmer et al.1994) or LepF1and LepR1 (Hebert, Penton, Burns, Janzen & Hallwachs 2004) and visualized ina 96-well E-Gel (Invitrogen Canada, Burlington, ON, Canada). The PCR mix included 6.25 mL of 10% trehalose, 1.25 mL 10 PCR buier, 0.625 mL (50mM) MgCl2,0.125 mL (10 mm) of each primer, 0.0625 mL (10mM) dNTPs, 0.0625 mL Platinum Aquaculture Research, 2009, 40, 973^979 doi:10.1111/j.1365-2109.2008.02159.x

Gold Nanoparticle Effects in Polymerase Chain Reaction: Favoring of Smaller Products by Polymerase Adsorption

Gold nanoparticles were recently reported to reduce the formation of nonspecific products in polymerase chain reaction (PCR) at remarkably low temperatures, with hypothesized mechanisms including adsorption of DNA and heat-transfer enhancement. In contrast to these reports, we report that gold nanoparticles do not enhance the specificity of PCR but rather suppress the amplification of longer products while favoring amplification of shorter products, independent of specificity. Gold nanoparticles bearing a self-assembled monolayer of hexadecanethiol did not affect PCR, suggesting that surface interactions play an essential role. This role was further confirmed by experiments in which a similar effect on PCR was observed for the same total surface area of particles over a 100-fold range of per-particle surface area. The effect was seen with Taq and Tfl polymerases but not with Vent polymerase, and the effects of nanoparticles can be reversed by increasing the polymerase concentration or by adding bovine serum albumin (BSA). Transient high-temperature nanoparticle pre-exposure of PCR mix containing polymerase but not template or primers, followed by nanoparticle removal, modified subsequent nanoparticle-free PCR. Interaction between polymerase and gold nanoparticles was confirmed by changes in nanoparticle absorption spectrum and electrophoretic mobility in the presence of polymerase. Taken together, these results suggest that the nanoparticles nonspecifically adsorb polymerase, thus effectively reducing polymerase concentration.

Molecular, Serological And Microbiological Profiling Evidence Of The Formit Transmission Of <i>Escherichia Coli</i> O157: Nm To A 1-Year Old Nigerian Child

Transmission of Escherichia Coli O157 by the formit route is under reported since interest in E. Coli O157 is usually arisen during outbreak situations which are mostly linked to consumption of food of bovine origin (Armstrong et al., 1996; Fukushima et al., 1999 and Allison et al., 2000). In December 2005 while working on the isolation of E. Coli O157 from cattle herds in Zaria, Kaduna State, a 1– year old child of a member of the team came down with a mild watery diarrhea. He was not presented to any Physician, since he was observed to be alert, active and with a normal appetite. However, stool specimens were collected from the child on days 2 and 3 of the diarrhea, (which was self limiting on day 3). Stool specimens were also collected from all the five members of his family. All items that the boy had contact with including a laboratory coat, bunch of keys and shoes were swabbed. Finally samples of all the boy's food and drinks were taken. Microbiological, Serological and Polymerase Chain Reaction (PCR) Profiling Assays. l the samples were cultured on Sorbitol - MacConkey (SMAC) agar, after enrichment using Modified Tryptone Soya Broth (MTSB) supplemented with Novobiocin (Oxoid Ltd, Basingstoke, England). All colourless colonies on SMAC agar from the samples were biochemically confirmed as E. coli by the methods described by (Cowan, 1981) and serologically as E. Coli O157:NM using the Remel Wellcolex E. coli O157:H7 kit (Remel Europe Ltd, Kent UK). Shigatoxin profiling was done using the VTEC-RPLA kit (Oxoid Ltd, Basingstoke England). Antimicrobial resistance profiling was carried out by the method of Bauer et al. (1966) using 16 antimicrobial agents: Ampicillin (AMP10), Ceftazidime (CAZ30), Cefuroxime (CXM30), Cephazolin (KZ30), ciprofloxacin (CIP5), compound Sulphonamides (S3300), gentamicin (CN10), Kanamycin (K30), Chloramphenicol (C30), Nalidixic acid (NA30), Neomycin (N10), Norfloxacin (NOR2), streptomycin (S10), Sulphamethoxazole (RL25), Co-trimoxazole (SXT25) and tetracycline (TE30) (Oxoid Ltd, Basingstoke, England). The assays were controlled using a control strain of E. Coli O157:H7 (ATCC 43895). Total DNA was isolated from 1 ml of brain heart infusion (BHI) broth culture grown overnight based on the method of Silhavy et al (1984). The Eae primer (Operon Ltd, Dusseldorf, Germany) was used (Germani et al., 1997). The PCR mix consisted of 2.50&#956l 10 x buffer, 1.50&#956l MgCl2, 0.50 dNTPs, eae-f 0.40&#956l, Taq polymerase (Ampli Taq Gold, Applied Biosystems, California, U.S.A.) 0.50&#956l, and 4.0&#956l of template DNA. The volume of the mix was adjusted to 25&#956l by addition of 15.2&#956l of nuclease free sterile water. DNA amplification was carried out in a Gene Amp PCR System 9700 Thermocycler (Applied Biosystems, California, U.S.A.) using an initial denaturation step at 94oC for 5 minutes, followed by 30 cycles of amplification with denaturation step at 94oC for 1 minute, annealing at 53oC for 1 minute, and extension at 72oC for 10 minutes and cooled to 4oC. Keywords : Escherichia Coli O157: NM, Formit, Transmission Nigerian Veterinary Journal Vol. 28 (3) 2007 pp. 53-55

A One-Step DNA PCR-based Method for the Detection of Mycobacterium tuberculosis Complex Grown on Lowenstein-Jensen Media

A simple, rapid, and sensitive direct colony polymerase chain reaction (PCR) method to detect Mycobacterium tuberculosis complex grown on Lowenstein-Jensen media is described. M. tuberculosis is killed by treating it for 2 hours with 70% ethanol. Whole Mycobacterium cells inactivated by ethanol are added to a PCR mix that is designed to amplify the IS6110 insertion sequence. All 44 isolates tested were positive by this method. Our results show that PCR can be performed directly on bacterial colonies without the need for DNA extraction before PCR. Moreover, inactivation of M. tuberculosis before DNA amplification reduces the potential exposure of workers to viable M. tuberculosis. The exclusion of DNA extraction and inactivation of colonies before PCR provide a safe and low-cost preparatory technique for PCR reaction compared with expensive conventional extraction protocols that are based on chemical and enzymatic lysis, especially for countries with limited resources.

PCR-based detection in a micro-fabricated platform

We present a novel, on-chip system for the electrokinetic capture of bacterial cells and their identification using the polymerase chain reaction (PCR). The system comprises a glass-silicon platform with a set of micro-channels, -chambers, and -electrodes. A platinum thin film resistor, placed in the proximity of the chambers, is used for temperature monitoring. The whole chip assembly is mounted on a Printed Circuit Board (PCB) and wire-bonded to it. The PCB has an embedded heater that is utilized for PCR thermal cycle and is controlled by a Lab-View program. Similar to our previous work, one set of electrodes on the chip inside the bigger chamber (0.6 microl volume) is used for diverting bacterial cells from a flowing stream into to a smaller chamber (0.4 nl volume). A second set of interdigitated electrodes (in smaller chamber) is used to actively trap and concentrate the bacterial cells using dielectrophoresis (DEP). In the presence of the DEP force, with the cells still entrapped in the micro-chamber, PCR mix is injected into the chamber. Subsequently, PCR amplification with SYBR Green detection is used for genetic identification of Listeria monocytogenes V7 cells. The increase in fluorescence is recorded with a photomultiplier tube module mounted over an epifluorescence microscope. This integrated micro-system is capable of genetic amplification and identification of as few as 60 cells of L. monocytogenes V7 in less than 90 min, in 600 nl volume collected from a sample of 10(4) cfu ml(-1). Specificity trials using various concentrations of L. monocytogenes V7, Listeria innocua F4248, and Escherichia coli O157:H7 were carried out successfully using two different primer sets specific for a regulatory gene of L. monocytogenes, prfA and 16S rRNA primer specific for the Listeria spp., and no cross-reactivity was observed.

Helicobacter pylori Gastritis in Children Is Associated With a Regulatory T-Cell Response

Helicobacter pylori infection in children infrequently causes gastroduodenal mucosal ulceration. Because H pylori induces T-cell dependent gastric inflammation in adults and T regulatory (Treg) cells suppress T-cell-dependent pathology, we evaluated gastric histopathology and Treg cell responses in H pylori-infected children and adults. Gastric tissue from 36 children and 79 adults with abdominal symptoms in Santiago, Chile, was evaluated prospectively for H pylori bacteria and histopathology using the Sydney classification and Treg responses using immunoassay, immunohistochemistry, and real-time polymerase chain reaction. Eighteen (50%) of the children and 51 (65%) of the adults were infected with H pylori. Children and adults were colonized with similar levels of H pylori. However, the level of gastritis in the children was reduced substantially compared with that of the adults (P < .05). Coincident with reduced gastric inflammation, the number of Treg cells and levels of Treg cytokines (transforming growth factor [TGF]-beta1 and interleukin-10) were increased markedly in the gastric mucosa of H pylori-infected children compared with that of infected adults (P < .03 and < .05, respectively). Also, H pylori infection in the children was associated with markedly increased levels of gastric TGF-beta1 and interleukin-10 messenger RNA. Importantly, gastric TGF-beta1 in H pylori-infected children localized predominantly to mucosal CD25(+) and Foxp3(+) cells, indicating a Treg source for the TGF-beta1. Gastric pathology is reduced and local Treg cell responses are increased in H pylori-infected children compared with infected adults, suggesting that gastric Treg cell responses down-regulate the inflammation and ulceration induced by H pylori in children.