A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

Background

Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations.

Methodology/Principal Findings

We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically.

Conclusions/Significance

The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control.     

Specific Dot-Immunobinding Assay for Detection and Enumeration of Thiobacillus ferrooxidans

A specific and very sensitive dot-immunobinding assay for the detection and enumeration of the bioleaching microorganism Thiobacillus ferrooxidans was developed. Nitrocellulose spotted with samples was incubated with polyclonal antisera against whole T. ferrooxidans cells and then in ¹²⁵I-labeled protein A or ¹²⁵I-labeled goat antirabbit immunoglobulin G; incubation was followed by autoradiography. Since a minimum of 10³ cells per dot could be detected, the method offers the possibility of simultaneous processing of numerous samples in a short time to monitor the levels of T. ferrooxidans in bioleaching operations.     

多孔板-MTT比色法测定植物抗菌成分对细菌的抑制活性

多孔板-MTT比色法测定植物抗菌成分对细菌抑制活性的步骤为：每孔加入浓度为10^6cfu／mL的供试菌液90灿,然后加入不同浓度的药液10μL,28℃暗培养24h后,每孔中加入5mg／mL的MTT溶液10μL,继续培养4h后加入10％二甲基亚砜100μL,振荡30min,在570nm处测定溶液的吸光值。采用以上方法,测定植物抗菌成分蓝桉醇对辣椒斑点病黄单胞菌（Xanthomonas vesicatoria）和枯草芽孢杆菌（Bacillus subtilis）的半抑制浓度（IC50）分别为0．158和0．395mg／mL,小檗碱对溶血葡萄球菌（Staphylococcus haemolyticus）的IC50为0．587mg／mL。结果表明,多孔板-MTT比色法能快速、微量地测定植物成分对细菌的抑制活性。     

A Rapid Colorimetric Assay for Determination of Leukocyte-Mediated Inhibition of Mycelial Growth of Candida albicans

A staining method with crystal violet (CV) was demonstrated to be useful for a simple, quick and objective assessment of in vitro growth inhibitory activity of leukocytes against Candida albicans cells. Candida cells incubated with murine neutrophils or macrophages for 14 hr in microwells were stained with CV and, after washing with 0.25% sodium dodecyl sulfate (SDS), treated with isopropanol containing HCl (0.04 N) to extract Candida cell-bound CV. Then the absorbance at 590 nm of the isopropanol extract was photometrically measured. The results showed that the photometrical absorbance was proportional to the amount of³H-glucose taken up by C. albicans cells, which reflected the number of viable Candida cells.     

A Colorimetric Assay for Cytokinin Oxidase

A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal andp-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.     

Colorimetric Assay for Lysine Decarboxylase in Escherichia coli

A new assay is described for lysine decarboxylase. It is rapid and reproducible in assaying large numbers of samples, a situation in which earlier methods were less convenient. The new method is valuable in the study of peptide fractions and amino acid mixtures which stimulate induction of lysine decarboxylase. It may be useful for work on enzyme structure and modification, genetics, and kinetics.     

Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Pseudomonas fluorescens Proteases in Ultrahigh-Temperature-Treated Milk

An inhibition enzyme-linked immunosorbent assay was developed to detect low levels of the proteases extracted from four strains of Pseudomonas fluorescens. The assay detected between 0.24 and 7.8 ng of protease per ml of ultrahigh-temperature-treated milk and could be completed within 6 h. It could be used as a framework for a test system for quantifying spoilage proteases in dairy products.     

A Multiplexed Microfluidic PCR Assay for Sensitive and Specific Point-of-Care Detection of Chlamydia trachomatis

Background

Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted diseases (STD) worldwide. While commercial nucleic acid amplification tests (NAAT) are available for Ct, none are rapid or inexpensive enough to be used at the point-of-care (POC). Towards the first Ct POC NAAT, we developed a microfluidic assay that simultaneously interrogates nine Ct loci in 20 minutes.

Methodology and Principal Findings

Endocervical samples were selected from 263 women at high risk for Ct STDs (∼35% prevalence). A head-to-head comparison was performed with the Roche-Amplicor NAAT. 129 (49.0%) and 88 (33.5%) samples were positive by multiplex and Amplicor assays, respectively. Sequencing resolved 71 discrepant samples, confirming 53 of 53 positive multiplex samples and 12 of 18 positive Amplicor samples. The sensitivity and specificity were 91.5% and 100%, and 62.4% and 95.9%, respectively, for multiplex and Amplicor assays. Positive and negative predictive values were 100% and 91%, and 94.1% and 68.6%, respectively.

Conclusions

This is the first rapid multiplex approach to Ct detection, and the assay was also found to be superior to a commercial NAAT. In effect, nine simultaneous reactions significantly increased sensitivity and specificity. Our assay can potentially increase Ct detection in globally diverse clinical settings at the POC.     

酶放大镧系元素发光法测定碱性磷酸酶

报告了应用酶放大镧系元素发光法测定碱性磷酸酶的方法.应用5-氟水杨酸磷酸酯作为酶底物,并对方法学中的多种因素进行了最佳比.用甲基硅油(I)改进了信/噪比的稳定性.方法的灵敏度为4 U/L.精密度为10%.精密度为10%的浓度范围是2.00～3.00×10² U/L.测量值的相对误差＜10%.测定了血清中的碱性磷酸酶浓度,回收率为93%～95%.     

Enumeration of Acetogens by a Colorimetric Most-Probable-Number Assay

Anaerobic O demethylation by acetogenic bacteria often is the first step in the mineralization of methoxylated aromatic compounds in anoxic environments. In this reaction, an ether bond is cleaved and the resulting methyl group is metabolized via the acetyl coenzyme A pathway (acetogenesis). Anaerobic O demethylation was used to assess acetogen populations. Environmental samples were diluted in anaerobic medium containing a methoxylated aromatic substrate (vanillate) and titanium(III), and acetogen titers were estimated by the most-probable-number (MPN) method. Complex formation between Ti(III) and vicinal hydroxyl groups of the aromatic products of anaerobic O demethylation results in the development of a yellow color in the medium, which can be detected by eye and monitored spectrophotometrically. High-performance liquid chromatography analysis of the yellow MPN tubes showed that they contained the product of anaerobic O demethylation of vanillate (protocatechuate). This assay was used to enumerate O-demethylating acetogen populations in environmental samples.     

Trichlorfon-Induced Inhibition of Nitrate and Ammonium Uptake in Cyanobacteria

The possibility that the primary effect of the toxic insecticidetrichlorfon is an inhibition of nitrate uptake in cyanobactenahas been investigated. A drastic reduction in the rate of uptakeis detected 3 h after the addition of the insecticide to batchcultures of nabaena PCC 7119. The dose-response curves indicatea relationship between the degree of inhibition of nitrate uptakeand the reduction of chlorophyll content and growth. Nitratereductase (ferredoxin : nitrate reductase, EC 1.7.99.4 [EC] ) activityis also lowered as a result of insecticide action. When AnabaenaPCC 7119 cells are grown with ammonium as a source of combinednitrogen, trichlorfon reduces the rate of ammonium uptake. Therate of uptake of both nitrate and ammonium is restored uponwashing the cells. Ultrastructural analysis of Anabaena nitrate-growncells shows that trichlorfon does not damage thylakoid membranes,but brings about the accumulation of enlarged cyanophycin granulesand the increase of carboxysome number. Nitrate uptake rateand chlorophyll and phycobiliprotein contents are also reducedby insecticide treatment in the cyanobacteria SynechococcusUAM 211, GloeothecePCC 6501, Plectonema calothricoides, NostocUAM 205 and Chlorogloeopsis PCC 6912. These results are consistentwith the inhibition of nitrate uptake due to weak adsorptionof trichlorfon to the plasmalemma being the main effect of theinsecticide on cyanobacterial metabolism. Key words: Nitrate uptake, cyanobacteria, Anabaena, ammonium uptake, trichlorfon     

Colorimetric Assay System for Screening Antiviral Compounds against Hepatitis B Virus

A highly sensitive, rapid, and accurate assay system was developed for the in vitro evaluation of anti-hepatitis B virus (anti-HBV) agents. Chronic HBV-producing HB611 cells were used in combination with immunoaffinity purification, polymerase chain reaction (PCR), and hybrid capture detection. HB611 cells were incubated with putative anti-HBV agents for 7 days in 96-well microtiter plates. HBV was purified from HB611 cell culture media using immunoaffinity purification. The HBV DNA was extracted, amplified with PCR, and assayed using a hybrid capture colorimetric method. This assay provided quantitative detection of extracellular HBV DNA from 25 μl of cell culture media. Using the colorimetric method, we found that 50% effective concentration levels of several known anti-HBV agents (HPMPA, PMEDAP, PMEA and others) were similar to those reported in studies using Southern blot analysis. These results demonstrate that this new and easily automated colorimetric assay system can be used for the rapid and accurate assessment of anti-HBV compound selectivity.     

Cationic Surfactant-Based Colorimetric Detection of Plasmodium Lactate Dehydrogenase,a Biomarker for Malaria,Using the Specific DNA Aptamer

A simple, sensitive, and selective colorimetric biosensor for the detection of the malarial biomarkers Plasmodium vivax lactate dehydrogenase (PvLDH) and Plasmodium falciparum LDH (PfLDH) was demonstrated using the pL1 aptamer as the recognition element and gold nanoparticles (AuNPs) as probes. The proposed method is based on the aggregation of AuNPs using hexadecyltrimethylammonium bromide (CTAB). The AuNPs exhibited a sensitive color change from red to blue, which could be seen directly with the naked eye and was monitored using UV-visible absorption spectroscopy and transmission electron microscopy (TEM). The reaction conditions were optimized to obtain the maximum color intensity. PvLDH and PfLDH were discernible with a detection limit of 1.25 pM and 2.94 pM, respectively. The applicability of the proposed biosensor was also examined in commercially available human serum.     

Effect of Aluminum Phosphate on Alkaline Phosphatase Activity of Polyurethane Foam Immobilized Cyanobacteria

The impact of insoluble phosphorus such as aluminum and rock phosphate on alkaline phosphatase activity of polyurethane foam immobilized cyanobacteria was assessed. Polyurethane foam immobilized Nodularia recorded the highest alkaline phosphatase activity of 9.04 (m. mol p-nitrophenol released h^–1 mg^–1 protein) in vitro. A higher concentration of aluminum phosphate was recorded a 25% reduction in alkaline phosphatase activity, ammonia content, and available phosphorus in culture filtrate of polyurethane foam immobilized cyanobacteria. In general, immobilized cyanobacteria exhibited a higher alkaline phosphatase activity in rock phosphate than aluminum phosphate.     

ELISA法检测流行性腮腺炎病毒感染特异性IgM抗体的研究

用流行性腮腺炎(流腮)病毒Enders株接种鸡胚尿囊腔培养,尿囊液经聚乙二醇6000处理制备流腮病毒抗原,用ELISA法检测流腮患者血清中特异性IgM抗体,其敏感性,特异性、重复性和稳定性都很高。 79份流腮患者血清,检出特异性IgM72份,阳性率为91％,32例非流腮患者IgM全部阴性、两者有极显著差异(P<0.01)。 10份血清作血清倍比稀释至1∶3200测IgM仍全部阳性,1∶6400稀释仅1例阴性,1∶12800稀释5例中仍有2例阳性。 10份血清作流腮抗原特异性抗体阻断试验,光密度抑制率均大于50％,平均为87％,10份标本作2-ME和SPA阻断后检测IgM抗体,结果2-ME阻断标本全部阴转,而SPA阻断标本仍阳性,证实所检测为流腮特异性抗体。 24份标本2次重复检测流腮IgM,其阴、阳性结果一致,这期间抗原放4℃ 1个月,提示抗原的稳定性和方法的重复性都很好。本方法敏感性明显高于血凝抑制试验,其阳性率分别为91％和61％,两者有显著差异。而且所用试剂简单经济,操作简便,快速,适用于临床早期诊断,易于广泛推广应用。