Evaluation and improvement of LAMP assays for detection of Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157

Abstract: Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157 are the causative agents of human diseases, and LAMP assays have been developed for detection of the seven leading STEC serogroups.Objective: To evaluate existing LAMP assays for detection of the seven STEC serogroups, if necessary, to improve these assays and to promote their practical application.Methods: Pure DNA extract from 23 strains reserved in our lab was used to evaluate the existing LAMP assays. The existing LAMP assays were modified via adding 1% tetramethylene sulfoxide and 5% dimethyl sulfoxide as well as optimizing reaction temperature.Results: The detection limit of the modified LAMP assays was 0.1-1 pg per reaction, equivalent to 25-250 cfu per reaction, the non-specific amplification can completely be eliminated with optimal amplifying temperature, and the modified LAMP assays can specifically and sensitivly amplify targeted O serogroup-specific gene (wzx or wzy) of any concerned Escherichia coli serogroup as commercial kit Isothermal Master Mix did.Conclusion: In conclusion, the LAMP assays were highly susceptible to non-specific amplification caused by primer dimers, and these modified methods were free of non-specific amplification and can rapidly and reliably detect the seven major Shiga toxin-producing E. coli serogroups.Keywords: Loop-mediated Isothermal Amplification (LAMP), toxin-producing Escherichia coli serogroups, non-specific amplification, tetramethylene sulfoxide, dimethyl sulfoxide

Advance in Tribology Study of Polyelectrolyte Multilayers

This review introduced the preparation and structural characterization of polyelectrolyte multilayers in recent years and also summarized the tribology research progress of the polyelectrolyte multilayers, including tribological properties, surface adhesion characteristics, and wear resistance properties. Statistics analysis indicated that nanoparticles‐doped polyelectrolyte multilayers present better friction and wear performance than pristine polyelectrolyte multilayers. Furthermore, the in situ growth method resulted in improved structural order of nanoparticles composite molecular deposition film. In situ nanoparticles not only reduced the molecular deposition film surface adhesion force and friction force but also significantly improved the life of wear resistance. That was due to the nanoparticles that possessed a good load‐carrying capacity and reduced the mobility of the polymer‐chain segments, which can undergo reversible shear deformation. Based on this, further research direction of in situ nanoparticles molecular deposition film was proposed

Research Progress of the Drill String Hardbanding Materials

Among anti-wear technologies, hardbanding is the most effective measure to reduce the wear of drill pipe and casing. Many new hardbanding materials are introduced constantly, and the varieties of hardbandings are becoming more and more abundant. In this paper, the research history and status quo of the hardbanding materials in tool joint are reviewed. And the advantages and disadvantages of all kinds of wear-resisting materials are introduced. Finally, the development orientation in hardbanding materials in drill joint was pointed out

Evaluation and improvement of LAMP assays for detection of Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157.

Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157 are the causative agents of human diseases, and LAMP assays have been developed for detection of the seven leading STEC serogroups. Objective: To evaluate existing LAMP assays for detection of the seven STEC serogroups, if necessary, to improve these assays and to promote their practical application. Methods: Pure DNA extract from 23 strains reserved in our lab was used to evaluate the existing LAMP assays. The existing LAMP assays were modified via adding 1% tetramethylene sulfoxide and 5% dimethyl sulfoxide as well as optimizing reaction temperature. Results: The detection limit of the modified LAMP assays was 0.1-1 pg per reaction, equivalent to 25-250 cfu per reaction, the non-specific amplification can completely be eliminated with optimal amplifying temperature, and the modified LAMP assays can specifically and sensitivly amplify targeted O serogroup-specific gene (wzx or wzy) of any concerned Escherichia coli serogroup as commercial kit Isothermal Master Mix did. Conclusion: In conclusion, the LAMP assays were highly susceptible to non-specific amplification caused by primer dimers, and these modified methods were free of non-specific amplification and can rapidly and reliably detect the seven major Shiga toxin-producing E. coli serogroups

Characteristics of cadmium accumulation and tolerance in apple plants grown in different soils

Cadmium (Cd) is a nonessential element and highly toxic to apple tree. However, Cd accumulation, translocation and tolerance in apple trees planted in different soils remain unknown. To investigate soil Cd bioavailability, plant Cd accumulation, physiological changes as well as gene expression patterns in apple trees grown in five different soils, ‘Hanfu’ apple seedlings were planted in orchard soils collected from Maliangou village (ML), Desheng village (DS), Xishan village (XS), Kaoshantun village (KS) and Qianertaizi village (QT), and subjected to 500 μM CdCl2 for 70 d. Results showed that soils of ML and XS had higher content of organic matter (OM), clay and silt, and cation exchange capacity (CEC) but lower sand content than the other soils, thereby reduced Cd bioavailability, which could be reflected by lower concentrations and proportions of acid-soluble Cd but higher concentrations and proportions of reducible and oxidizable Cd. The plants grown in soils of ML and XS had relatively lower Cd accumulation levels and bio-concentration factors than those grown in the other soils. Excess Cd reduced plant biomass, root architecture, and chlorophyll content in all plants but to relatively lesser degree in those grown in soils of ML and XS. The plants grown in soils of ML, XS and QT had comparatively lower reactive oxygen species (ROS) content, less membrane lipid peroxidation, and higher antioxidant content and enzyme activity than those grown in soils of DS and KS. Transcript levels of genes regulating Cd uptake, transport and detoxification such as HA11, VHA4, ZIP6, IRT1, NAS1, MT2, MHX, MTP1, ABCC1, HMA4 and PCR2 displayed significant differences in roots of plants grown in different soils. These results indicate that soil types affect Cd accumulation and tolerance in apple plants, and plants grown in soils with higher OM content, CEC, clay and silt content and lower sand content suffer less Cd toxicity

The impact of intraarterial, intravenous, and combined tirofiban on endovascular treatment for acute intracranial atherosclerotic occlusion

Background and purposeAdjunctive tirofiban administration in patients undergoing endovascular treatment (EVT) for acute large vessel occlusion (LVO) has been investigated in several studies. However, the findings are conflict. This study aimed to compare the effect of different administration pathways of tirofiban on patients undergoing EVT for acute LVO with intracranial atherosclerotic disease (ICAD).MethodsPatients were selected from the ANGEL-ACT Registry (Endovascular Treatment Key Technique and Emergency Workflow Improvement of Acute Ischemic Stroke: A Prospective Multicenter Registry Study) and divided into four groups: intra-arterial (IA), intravenous (IV), and intra-arterial plus intravenous (IA+IV) and non-tirofiban. The primary outcome was 90-day ordinal modified Rankin Scale (mRS) score, and the secondary outcomes included the rates of mRS 0–1, 0–2, and 0–3 at 90-day, successful recanalization. The safety outcomes were symptomatic intracranial hemorrhage (sICH) and other safety endpoints. The multivariable logistic regression models adjusting for potential baseline confounders were performed to compare the outcomes. A propensity score matching (PSM) with a 1:1:1:1 ratio was conducted among four groups, and the outcomes were then compared in the post-matched population.ResultsA total of 502 patients were included, 80 of which were in the IA-tirofiban group, 73 in IV-tirofiban, 181 in (IA+IV)-tirofiban group, and 168 in the non-tirofiban group. The median (IQR) 90-day mRS score in the four groups of IA, IV, IA+IV, and non-tirofiban was, respectively 3(0–5) vs. 1(0–4) vs. 1(0–4) vs. 3(0–5). The adjusted common odds ratio (OR) for 90-day ordinal modified Rankin Scale distribution with IA-tirofiban vs. non-tirofiban was 0.77 (95% CI, 0.45–1.30, P = 0.330), with IV-tirofiban vs. non-tirofiban was 1.36 (95% CI, 0.78–2.36, P = 0.276), and with (IA+IV)-tirofiban vs. non-tirofiban was 1.03 (95% CI, 0.64–1.64, P = 0.912). The adjusted OR for mRS 0–1 and mRS 0–2 at 90-day with IA-tirofiban vs. non-tirofiban was, respectively 0.51 (95% CI, 0.27–0.98, P = 0.042) and 0.50 (95% CI, 0.26–0.94, P = 0.033). The other outcomes of each group were similar with non-tirofiban group, all P was >0.05. After PSM, the common odds ratio (OR) for 90-day ordinal modified Rankin Scale distribution with IA-tirofiban vs. non-tirofiban was 0.41 (95% CI, 0.18–0.94, P = 0.036), and the OR for mRS 0–1 and mRS 0–2 at 90-day with IA-tirofiban vs. non-tirofiban was, respectively 0.28 (95% CI, 0.11–0.74, P = 0.011) and 0.25 (95% CI, 0.09–0.67, P = 0.006).ConclusionsIntra-arterial administration of tirofiban was associated with worse outcome than non-tirofiban, which suggested that intra-arterial tirofiban had a harmful effect on patients undergoing EVT for ICAD-LVO.Clinical trial registrationhttp://www.clinicaltrials.gov, Unique identifier: NCT03370939

Detection of Cassava Component in Sweet Potato Noodles by Real-Time Loop-mediated Isothermal Amplification (Real-time LAMP) Method

Sweet potato (Ipomoea batatas) noodles are a traditional Chinese food with a high nutritional value; however, starch adulteration is a big concern. The objective of this study was to develop a reliable method for the rapid detection of cassava (Manihot esculenta) components in sweet potato noodles to protect consumers from commercial adulteration. Five specific Loop-mediated Isothermal Amplification (LAMP) primers targeting the internal transcribed spacer (ITS) of cassava were designed, genomic DNA was extracted, the LAMP reaction system was optimized, and the specificity of the primers was verified with genomic DNA of cassava, Ipomoea batatas, Zea mays, and Solanum tuberosum; the detection limit was determined with a serial dilution of adulterated sweet potato starch with cassava starch, and the real-time LAMP method for the detection of the cassava-derived ingredient in sweet potato noodles was established. The results showed that the real-time LAMP method can accurately and specifically detect the cassava component in sweet potato noodles with a detection limit of 1%. Furthermore, the LAMP assay was validated using commercial sweet potato noodle samples, and results showed that 57.7% of sweet potato noodle products (30/52) from retail markets were adulterated with cassava starch in China. This study provides a promising solution for facilitating the surveillance of the commercial adulteration of sweet potato noodles from retail markets

Effect of internal primer–template mismatches on loop-mediated isothermal amplification

The technique of loop-mediated isothermal amplification (LAMP) utilizes 4 (5 or 6) primers targeting 6 (7 or 8) regions within a fairly small genome segment for amplification. This technique has a potential for greater specificity than two-primer methods, such as polymerase chain reaction. There are still no reports for primer–template mismatch of LAMP. In this study, a set of LAMP primers was designed, targeting the 16S–23S rRNA intergenic spacer region of Streptococcus dysgalactiae. The selectivity of the LAMP method was tested with 25 bacterial strains. There was a non-specific amplification when the genomic DNA of Streptococcus agalactiae was used as a template and it was indicated by a nucleotide basic local alignment search tool (BLAST) in GenBank. There were three false priming sites on backward inner primer and the internal primer–template mismatches extended the detection time from 21 min to 47 min. This study would be of great reference value for targeting sequence selection, primer design of LAMP and detection of antibiotic-resistant bacteria with LAMP

Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae

Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae are the three main pathogens causing bovine mastitis, with great losses to the dairy industry. Rapid and specific loop-mediated isothermal amplification methods (LAMP) for identification and differentiation of these three pathogens are not available. With the 16S rRNA gene and 16S-23S rRNA intergenic spacers as targets, four sets of LAMP primers were designed for identification and differentiation of S. dysgalactiae, S. uberis and S. agalactiae. The detection limit of all four LAMP primer sets were 0.1 pg DNA template per reaction, the LAMP method with 16S rRNA gene and 16S-23S rRNA intergenic spacers as the targets can differentiate the three pathogens, which is potentially useful in epidemiological studies

Detection of chicken adulteration in beef via ladder-shape melting temperature isothermal amplification (LMTIA) assay

AbstractThe ladder-shape melting temperature isothermal amplification (LMTIA) is a newly developed technique with advantages of simple mechanism, easy primer design, quick turn-around time, high specificity, high sensitivity and short target sequence compared to the loop-mediated isothermal amplification (LAMP) technique. The objective of this research was to establish the LMTIA assay for detection of chicken in beef. The LMTIA primers targeting the prolactin receptor gene of Gallus gallus were designed, the LMTIA reaction system was optimized, the specificity and the sensitivity of the LMTIA assay were determined. Our results showed that the LMTIA assay was able to specifically detect 100 ng genomic DNA of Gallus gallus, without detecting the 100 ng genomic DNA of Anas platyrhynchos, Felis catus, Sus scrofa, Canis lupus familiaris, Bos taurus or Capra hircus. The sensitivity of the LMTIA assay was 100 pg genomic DNA of G. gallus. Furthermore, the LMTIA assay was able to detect the chicken adulterated in beef with ≥0.1% (w/w) detection limit (LOD). This study holds a promise for facilitation of the surveillance of the commercial adulteration of chicken in beef