Effect of prophylactic caffeine in the treatment of apnea in very low birth weight infants: a meta-analysis.

OBJECTIVE: The purpose of this meta-analysis is to investigate the effect of prophylactic caffeine use in the treatment of apnea and other clinical outcomes in very low birth weight infants. METHODS: We searched PubMed, Embase, Web of Science, Scopus, EBSCO, CNKI, and Cochrane databases for all relevant studies up to May 20, 2022. The meta-analysis was carried out using Stata16.0 and RevMan5.4 software. RESULTS: Eleven randomized controlled trials were evaluated, including a total of 4375 very low birth weight infants. The results demonstrated that prophylactic caffeine use was linked with a significantly lower probability of AOP (OR 0.31, 95% CI: 0.19-0.49, p < .001), duration of mechanical ventilation and oxygen therapy when compared to the control group. It also reduced the incidence of BPD (OR 0.62, 95% CI: 0.54-0.71, p < .001), PDA (OR 0.49, 95% CI: 0.30-0.80, p = .005) and ROP (OR 0.76, 95% CI: 0.65-0.90, p = .001), without raising the risk of NEC, IVH and death before hospital discharge (p > .05). CONCLUSION: This meta-analysis confirmed the beneficial effects of prophylactic caffeine in preventing apnea of prematurity and improving clinical outcomes.

Molecular Epidemiology of Blastocystis in Confined Slow Lorises, Macaques, and Gibbons.

Blastocystis sp. is a common intestinal anaerobic parasite infecting non-human primates and many other animals. This taxon threatens the health of NHPs due to its high genetic diversity, impeding efforts to improve confined management and subsequent conservation practices. This study collected 100 and 154 fecal samples from captive macaques, gibbons, and slow lorises in the summer and winter, respectively. The Blastocystis infection, its gene subtypes, and its zoonotic potential based on small subunit ribosomal RNA (SSU rRNA) were analyzed. The prevalence of Blastocystis in the three primate genera was 57.79% (89/154) in the summer (2021) and 29.00% (29/100) in the winter (2020). Four zoonotic subtypes-ST1, ST2, ST3, and ST4-were identified. ST2 was the most prevalent subtype, suggesting that these animals may serve as reservoirs for pathogens of human Blastocystis infections. The macaques showed a more significant variation in Blastocystis infection between seasons than gibbons and slow lorises. The slow lorises in small cages and enclosure areas were potentially more infected by Blastocystis in the summer, indicating that inappropriate captive management may have detrimental effects on their health.

Efficacy of propylthiouracil in the treatment of pregnancy with hyperthyroidism and its effect on pregnancy outcomes: A meta-analysis.

BACKGROUND: Hyperthyroidism affects about 0.2%-2.7% of all pregnancies, and is generally treated with propylthiouracil (PTU). However, previous studies about the effects of propylthiouracil on maternal or foetal are contentious. OBJECTIVE: This meta-analysis was carried out to investigate the safety and efficacy of propylthiouracil during pregnancy. MATERIALS AND METHODS: PubMed, EBSCO, Embase, Scopus, Web of Science, Cochrane, CNKI, Wanfang and VIP database were searched from inception until August 31, 2021 for all available randomized controlled trials (RCTs) or cohort studies that evaluated the efficacy of propylthiouracil and its effects on pregnancy outcomes. Odds ratio (OR) and 95% confidence interval (CI) were used for binary variables, weighted mean difference (WMD) and 95% confidence interval (CI) were used for continuous variables. RevMan5.4 and Stata 16.0 were used for performing the meta-analysis. RESULTS: The researchers examined data from 13 randomized controlled trials and cohort studies involving 18948 infants. Congenital anomalies were not significantly associated with PTU in the pooled results (OR = 1.03, 95%CI: 0.84-1.25, P = 0.80, I2 = 40.3%). There were no statistically significant differences in neonatal hypothyroidism (OR = 0.55, 95%CI: 0.06-4.92, P = 0.593, I2 = 57.0%) or hepatotoxicity (OR = 0.34, 95%CI: 0.08-1.48, P = 0.151, I2 = 0.0%) exposed to PTU compared to the control group. The serum levels of FT3, FT4, TT3, and TT4 were significantly lower in the propylthiouracil group compared to the control group. CONCLUSION: This meta-analysis confirmed the beneficial effects of propylthiouracil treatment, namely the risks of adverse pregnancy outcomes were not increased, and it also proved PTU's efficacy in the treatment of pregnant women with hyperthyroidism. The findings supported the use of propylthiouracil during pregnancy with hyperthyroidism in order to improve clinical pregnancy outcomes in patients with thyroid dysfunction.

Non-invasive monitoring method for lower-leg compartment syndrome using a wireless sensor system and finite element analysis.

In this study, a non-invasive pressure monitoring system that is portable and convenient was designed for detecting compartment syndrome. The system combines a wireless module and smartphone, which aids in the achievement of mHealth objectives, specifically, the continuous monitoring of the compartment pressure in patients. A compartment syndrome detecting method using a wireless sensor system and finite element analysis is developed and verified with an in vitro lower-leg model by rapid prototyping. The sensor system is designed to measure a five point pressure variation from the outside of the lower leg and transmit the data to a smartphone via Bluetooth. The analysis model based on the finite element method is employed to calculate the change of pressure and volume inside the four compartments of the lower leg. The in vitro experimental results show that the non-invasive detecting method can monitor the compartment pressure and provide a warning for the occurrence of compartment syndrome if the compartment pressure is higher than 30 mmHg. Furthermore, the theoretical simulation of the real lower leg shows similar trends to those of the in vitro experiments and can promptly detect the occurrence of compartment syndrome. Measured pressure values exceeding 6.3, 2.7, and 2.8 kPa for the three sensors contacting the outside centers of the superficial posterior, anterior, and lateral compartments, respectively, can indicate that each compartment contains a pressure higher than 30 mmHg. These results can provide a warning for the risk of compartment syndrome of each compartment. In addition, the measured values from the three sensors contacting the superficial posterior compartment at the outside center, close to the tibia, and close to the lateral compartment exceeding 1.8, 0.7, and 0.7 kPa, respectively, can indicate the risk of deep posterior compartment syndrome.

Chinese newborn screening for the incidence of G6PD deficiency and variant of G6PD gene from 2013 to 2017.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common X-linked enzymopathies caused by G6PD gene variant. We aimed to provide the characteristics of G6PD deficiency and G6PD gene variant distribution in a large Chinese newborn screening population. We investigated the prevalence of G6PD in China from 2013 to 2017. Then, we examined G6PD activity and G6PD gene in representative Chinese birth cohort to explore the distribution of G6PD gene variant in 2016. We then performed multicolor melting curve analysis to classify G6PD gene variants in 10,357 neonates with activity-confirmed G6PD deficiency, and DNA Sanger sequencing for G6PD coding exons if hot site variants were not found. The screened population, organizations, and provinces of G6PD deficiency were increased from 2013 to 2017 in China. The top five frequency of G6PD gene variants were c.1376G>T, c.1388G>A, c.95A>G, c.1024C>T, and c.871G>A and varied in different provinces, with regional and ethnic features, and four pathogenic variant sites (c.152C>T, c.290A>T, c.697G>C, and c.1285A>G) were first reported. G6PD deficiency mainly occurs in South China, and the frequency of G6PD gene variant varies in different regions and ethnicities.

Parameters Affecting the Antimicrobial Properties of Cold Atmospheric Plasma Jet.

Using the Taguchi method to narrow experimental parameters, the antimicrobial efficiency of a cold atmospheric plasma jet (CAPJ) treatment was investigated. An L9 array with four parameters of CAPJ treatments, including the application voltage, CAPJ-sample distance, argon (Ar) gas flow rate, and CAPJ treatment time, were applied to examine the antimicrobial activity against Escherichia coli (E. coli). CAPJ treatment time was found to be the most influential parameter in its antimicrobial ability by evaluation of signal to noise ratios and analysis of variance. 100% bactericidal activity was achieved under the optimal bactericidal activity parameters including the application voltage of 8.5 kV, CAPJ-sample distance of 10 mm, Ar gas flow rate of 500 sccm, and CAPJ treatment time of 300 s, which confirms the efficacy of the Taguchi method in this design. In terms of the mechanism of CAPJ's antimicrobial ability, the intensity of hydroxyl radical produced by CAPJ positively correlated to its antimicrobial efficiency. The CAPJ antimicrobial efficiency was further evaluated by both DNA double-strand breaks analysis and scanning electron microscopy examination of CAPJ treated bacteria. CAPJ destroyed the cell wall of E. coli and further damaged its DNA structure, thus leading to successful killing of bacteria. This study suggests that optimal conditions of CPAJ can provide effective antimicrobial activity and may be grounds for a novel approach for eradicating bacterial infections.

Dysregulation of microRNA-657 influences inflammatory response via targeting interleukin-37 in gestational diabetes mellitus.

A number of studies have implicated that microRNAs (miRNAs) play a critical role in the development of gestational diabetes mellitus (GDM). However, the role of miR-657 in GDM remains vague up to date. We aim to investigate the modifying effect of miR-657 on GDM, which will provide new insight into the pathogenesis of GDM and may help to identify new diagnostic or therapeutic targets for GDM. Increased expression of miR-657 but decreased expression of interleukin-37 (IL-37) was observed in patients with GDM. Besides, negative association between miR-657 and IL-37 was demonstrated in this study. miR-657 could targetedly regulate IL-37 and enhance the proliferation of mononuclear macrophages. Moreover, miR-657 promoted the generation of inflammatory cytokines (IL-6 and tumor necrosis factor-α [TNF-α]) and activation of nuclear factor κB (NF-κB) in lipopolysaccharide-induced mononuclear macrophages, while its effect was significantly inhibited when exogenous recombinant IL-37 was administrated into cells. Accordingly, dysregulation of miR-657 contributes to the pathogenesis of GDM via IL-37/NF-κB signaling axis.

Photoperiodic diapause in a subtropical population of Aedes albopictus in Guangzhou, China: optimized field-laboratory-based study and statistical models for comprehensive characterization.

BACKGROUND: Aedes albopictus is among the 100 most invasive species worldwide and poses a major risk to public health. Photoperiodic diapause provides a crucial ecological basis for the adaptation of this species to adverse environments. Ae. albopictus is the vital vector transmitting dengue virus in Guangzhou, but its diapause activities herein remain obscure. METHODS: In the laboratory, yeast powder and food slurry were compared for a proper diapause determination method, and the critical photoperiod (CPP) was tested at illumination times of 11, 11.5, 12, 12.5, 13, and 13.5 h. A 4-parameter logistic (4PL) regression model was selected to estimate the CPP. In the field, the seasonal dynamics of the Ae. albopictus population, egg diapause, and hatching of overwintering eggs were investigated monthly, weekly, and daily, respectively. A distributed lag non-linear model (DLNM) was used to assess the associations of diapause with meteorological factors. RESULTS: In the laboratory, both the wild population and the Foshan strain of Ae. albopictus were induced to diapause at an incidence greater than 80%, and no significant difference (P > 0.1) was observed between the two methods for identifying diapause. The CPP of this population was estimated to be 12.312 h of light. In the field, all of the indexes of the wild population were at the lowest levels from December to February, and the Route Index was the first to increase in March. Diapause incidence displayed pronounced seasonal dynamics. It was estimated that the day lengths of 12.111 h at week2016, 43 and 12.373 h at week2017, 41 contributed to diapause in 50% of the eggs. Day length was estimated to be the main meteorological factor related to diapause. CONCLUSIONS: Photoperiodic diapause of Ae. albopictus in Guangzhou of China was confirmed and comprehensively elucidated in both the laboratory and the field. Diapause eggs are the main form for overwintering and begin to hatch in large quantities in March in Guangzhou. Furthermore, this study also established an optimized investigation system and statistical models for the study of Ae. albopictus diapause. These findings will contribute to the prevention and control of Ae. albopictus and mosquito-borne diseases.

Hollow carbon dots labeled with FITC or TRITC for use in fluorescent cellular imaging.

Hollow carbon dots (HCDs) were prepared by a solvothermal method and conjugated to either tetramethyl rhodamine isothiocyanate (TRITC) or fluorescein-5-isothiocyanate (FITC). This resulted in HCDs with bright red or green fluorescence, with excitation/emission peaks at 550/580 and 491/520 nm, respectively. The nanocomposites are well water-soluble, remarkably photostable and biocompatible. In addition, the fluorescence of the composites is more stable in a reactive oxygen environment than the free dyes. Confocal images indicate that the nanoparticles quickly enter A549 cells and mainly accumulate in the cytoplasm. The wavelength of functionalized HCDs can be regulating via coupling the HCDs to different dyes. These results demonstrate that these composite materials can be very promising reagents for biological labeling and imaging. Graphical abstract Schematic of the preparation of hollow carbon dots conjugated to tetramethyl rhodamine isothiocyanate (RHCDs) by solvothermal method. The material is water-soluble, remarkably photostable and biocompatible. It was applied to cellular labeling and imaging.

Repression of Rgg But Not Upregulation of LacD.1 in emm1-type covS Mutant Mediates the SpeB Repression in Group A Streptococcus.

CovR/CovS is an important two-component regulatory system in human pathogen group A Streptococcus (GAS). Epidemiological studies have shown that inactivation of the sensor kinase CovS is correlated with invasive clinical manifestations. The phosphorylation level of response regulator CovR decreases dramatically in the absence of CovS, resulting in the derepression of virulence factor expression and an increase in bacterial invasiveness. Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease and is negatively regulated by CovR; however, the expression of SpeB is almost completely repressed in the covS mutant. The present study found that in the emm1-type A20 strain, non-phosphorylated CovR acts as a transcriptional repressor for SpeB-positive regulator Rgg. In addition, the expression of Rgg-negative regulator LacD.1 is upregulated in the covS mutant. These results suggest that inactivation of Rgg in the covS mutant would directly mediate speB repression. The current study showed that overexpression of rgg but not inactivation of lacD.1 in the covS mutant partially restores speB expression, indicating that only rgg repression, but not lacD.1 upregulation, contributes to the speB repression in the covS mutant.

[Diapause of Aedes albopictus and the Related Molecular Mechanisms].

Aedes albopictus is an important transmitting vector of a wide range of arboviruses such as Dengue virus and Chikungunya virus. The rapid and aggressive global spread of Aedes albopictus contributes to the rapid expansion of Dengue virus. The diapause of Aedes albopictus provides a crucial ecological basis for its adaptation to a wide range of climatic and geographical conditions. This review gives an update on research progress on diapause of Aedes albopictus and the related molecular mechanisms.

Study on erythrosine-phen-Cd(II) systems by resonance Rayleigh scattering, absorption spectra and their analytical applications.

In pH 7.0-8.0 KH2PO4-Na2HPO4 buffer solution, Cd(II) reacted with 1,10-phenanthroline to form chelate cation [Cd(phen)3]2+, which further reacted with anion of erythrosine to form ternary ion-association complex through electrostatic attraction and hydrophobic effect. This process could result in remarkable absorption spectra change and produce obvious fading reaction at 528 nm. Absorbance change (ΔA) of system was directly proportional to the concentration of Cd(II). Hereby, a highly sensitive spectrophotometric method for the determination of Cd(II) was established. The molar absorption coefficient was 2.29×10(5) L mol(-1) cm(-1) and the detection limit of Cd(II) was 26.5 ng mL(-1). Furthermore, the resonance Rayleigh scattering (RRS) of this system with two peaks located at 371 and 590 nm enhanced significantly, and second-order scattering (SOS) and frequence doubling scattering (FDS) of this system changed notably at 640 and 350 nm, respectively. Under the optimum conditions, the scattering intensities (ΔIRRS, ΔIDWO-RRS, ΔISOS and ΔIFDS) had good linear relationship with the concentration of Cd(II) in certain ranges. The detection limits of Cd(II) were 1.27 ng mL(-1), 1.39 ng mL(-1), 4.03 ng mL(-1), 5.92 ng mL(-1) and 14.7 ng mL(-1) for dual-wavelength overlapping resonance Rayleigh scattering (DWO-RRS), RRS (371 nm), RRS (590 nm), SOS and FDS, respectively. In addition, the suitable reaction conditions and effects of coexisting substances were investigated. The methods had been successfully applied to the determination of Cd(II) in environmental water samples. The recovery range was between 93.0% and 103.0% and the relative standard deviation (RSD) was between 2.5% and 4.3%. The results were in agreement with those obtained from atomic absorption spectroscopy.

Validation of a commercial insulated isothermal PCR-based POCKIT test for rapid and easy detection of white spot syndrome virus infection in Litopenaeus vannamei.

Timely pond-side detection of white spot syndrome virus (WSSV) plays a critical role in the implementation of bio-security measures to help minimize economic losses caused by white spot syndrome disease, an important threat to shrimp aquaculture industry worldwide. A portable device, namely POCKIT™, became available recently to complete fluorescent probe-based insulated isothermal PCR (iiPCR), and automatic data detection and interpretation within one hour. Taking advantage of this platform, the IQ Plus™ WSSV Kit with POCKIT system was established to allow simple and easy WSSV detection for on-site users. The assay was first evaluated for its analytical sensitivity and specificity performance. The 95% limit of detection (LOD) of the assay was 17 copies of WSSV genomic DNA per reaction (95% confidence interval [CI], 13 to 24 copies per reaction). The established assay has detection sensitivity similar to that of OIE-registered IQ2000™ WSSV Detection and Protection System with serial dilutions of WSSV-positive Litopenaeus vannamei DNA. No cross-reaction signals were generated from infectious hypodermal and haematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), and hepatopancreatic parvovirus (HPV) positive samples. Accuracy analysis using 700 L. vannamei of known WSSV infection status shows that the established assayhassensitivity93.5% (95% CI: 90.61-95.56%) and specificity 97% (95% CI: 94.31-98.50%). Furthermore, no discrepancy was found between the two assays when 100 random L. vannamei samples were tested in parallel. Finally, excellent correlation was observed among test results of three batches of reagents with 64 samples analyzed in three different laboratories. Working in a portable device, IQ Plus™ WSSV Kit with POCKIT system allows reliable, sensitive and specific on-site detection of WSSV in L. vannamei.

Detection of Salmonella in chicken meat by insulated isothermal PCR.

Consumption of Salmonella-contaminated foods, such as poultry and fresh eggs, is known to be one of the main causes of salmonellosis. Conventional PCR methods, including real-time PCR for rapid detection of Salmonella, in general require skilled technicians and costly instruments. A recently developed novel convective PCR, insulated isothermal PCR (iiPCR), is carried out in polycarbonate capillary tubes. In this study, we designed TaqMan probes and PCR primers based on the yrfH gene encoding a heat shock protein for the iiPCR detection of Salmonella in chicken meat samples. The TaqMan probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine at the 5' and 3' ends, respectively. The PCR amplicon was 133 bp. A typical run of this iiPCR assay was completed within 1 h. Specific PCR products were obtained for 148 strains representing 49 serotypes of Salmonella tested. Under the same conditions, false-positive results were not obtained for 98 non-Salmonella strains tested, including strains of Enterobacteriaceae closely related to Salmonella. For chicken meat samples, with a 5-h enrichment step Salmonella at as low as 10° CFU/g of poultry meat could be detected. Because the amplification signals from the probes are detectable at 520 nm, identification of the PCR products by gel electrophoresis is not required. Compared with conventional PCR, the iiPCR system requires less expertise and provides an economical, reliable, and rapid tool for result interpretation. Detection results can be obtained within 8 h, including the enrichment and DNA extraction steps.

Visual detection of prion protein based on color complementarity principle.

Two complementary colors mixed in a proper proportion will produce a neutral color in the color theory. A novel colorimetric method on basis of the color complementarity principle has been well-established to detect recombinant prion protein (rPrP). We found that a colorless solution appeared after mixing orange CdTe quantum dots (QDs) with green-blue malachite green (MG) because of color complementarity. After the addition of rPrP into the mixed solution, the color changed from colorless to green-blue because rPrP could induce the aggregation of QDs, rapidly. And it could be observed by naked eyes. Based on this phenomenon, we developed a simple assay for visual detection of rPrP. At the same time, we obtained excellent correlation between absorption and concentrations of rPrP from 1 nmol L(-1) to 78 nmol L(-1) with the limit of detection of 0.3 nmol L(-1) (3σ). Moreover, it can be applied to determine rPrP in human serum successfully. Importantly, this assay possesses the advantages of simplicity, rapidity, sensitivity, and selectivity, and shows the potential in the clinical diagnostic test of early prion disease and provides the possibility of preventing the spread of prion diseases.

Development of TaqMan probe-based insulated isothermal PCR (iiPCR) for sensitive and specific on-site pathogen detection.

Insulated isothermal PCR (iiPCR), established on the basis of Ralyeigh-Bénard convection, is a rapid and low-cost platform for nucleic acid amplification. However, the method used for signal detection, namely gel electrophoresis, has limited the application of iiPCR. In this study, TaqMan probe-based iiPCR system was developed to obviate the need of post-amplification processing. This system includes an optical detection module, which was designed and integrated into the iiPCR device to detect fluorescent signals generated by the probe. TaqMan probe-iiPCR assays targeting white spot syndrome virus (WSSV) and infectious myonecrosis virus were developed for preliminary evaluation of this system. Significant elevation of fluorescent signals was detected consistently among positive iiPCR reactions in both assays, correlating with amplicon detection by gel electrophoresis analysis. After condition optimization, a threshold value of S/N (fluorescent intensity(after)/fluorescent intensity(before)) for positive reactions was defined for WSSV TaqMan probe-iiPCR on the basis of 20 blank reactions. WSSV TaqMan probe-iiPCR generated positive S/Ns from as low as 10(1) copies of standard DNA and lightly infected Litopenaeus vannamei. Compared with an OIE-certified nested PCR, WSSV TaqMan probe-iiPCR showed a sensitivity of 100% and a specificity of 96.67% in 120 WSSV-free or lightly infected shrimp samples. Generating positive signals specifically and sensitively, TaqMan probe-iiPCR system has a potential as a low-cost and rapid on-site diagnostics method.

Detection of white spot syndrome virus by polymerase chain reaction performed under insulated isothermal conditions.

Aiming to develop a rapid, low-cost, and user-friendly system for the diagnosis of white spot syndrome virus (WSSV), a PCR assay performed in capillary tubes under insulated isothermal conditions (iiPCR assay) was established on the basis of Rayleigh-Benard convection. WSSV amplicons were generated reproducibly within 30 min from a target sequence-containing plasmid in an iiPCR device, in which a special polycarbonate capillary tube (R-tube™) was heated isothermally by a copper ring attached to its bottom and shielded by a thermal baffle around its upper half. Furthermore, WSSV-specific amplicons were produced from nucleic acid extracts of WSSV-infected Penaeus vannamei in the WSSV iiPCR assay, with sensitivity comparable to that of an OIE-certified commercial nested PCR kit (IQ2000™ WSSV Detection and Prevention System). Specificity of the WSSV iiPCR assay was demonstrated as no amplicons were generated from shrimp genomic DNA, and IHHNV, MBV, and HPV DNA. iiPCR has a potential as a low-cost method for sensitive, specific and rapid detection of pathogens.

A thermally baffled device for highly stabilized convective PCR.

Rayleigh-Bénard convective PCR is a simple and effective design for amplification of DNA. Convective PCR is, however, extremely sensitive to environmental temperature fluctuations, especially when using small- diameter test tubes. Therefore, this method is inherently unstable with limited applications. Here, we present a convective PCR device that has been modified by adding thermal baffles. With this thermally baffled device the influence from fluctuations in environmental temperature were significantly reduced, even in a wind tunnel (1 m/s). The thermally baffled PCR instrument described here has the potential to be used as a low-cost, point-of-care device for PCR-based molecular diagnostics in the field.

Expression, purification, and characterization of protective MPT64 antigen protein and identification of its multimers isolated from nontoxic Mycobacterium tuberculosis H37Ra.

MPT64, a secreted protein of Mycobacterium tuberculosis (MTB), stimulates the immune reactions within cells and is a protective antigen that is lost by the bacilli Calmette-Guérin (BCG) vaccine during propagation. To minimize the toxicity caused by MTB, we used the MPT64 gene encoded by nontoxic H37Ra MTB to carry out genetic expansion via polymerase chain reaction and gene clone MPT64. The plasmid DNA encoded MPT64 was expressed at 20°C for 22 H, and a large quantity of MPT64 was obtained. In the absence of urea, MPT64 multimers with subunits being covalently connected via disulfide bonds were detected by Western blot showing strong protein-protein interactions, as evidenced by the formation of MPT64 tetramers. Finally, with urea of decreasing concentrations, we refolded MPT64 purified in the presence of urea and determined its secondary structures using circular dichroism. MPT64 was found to contain 2.2% α-helix, 50.9% ß-sheet, 19.5% turn, and 27.4% random coil. The molecular weight of MPT64 was determined by a matrix-assisted laser desorption ionization-time of flight mass spectrometer and found to be 23,497 Da, very close to the theoretical molecular weight of MPT64. The results presented here provide a sound basis for future biochemical and biophysical studies of MPT64 or any other proteins encoded by nontoxic H37Ra MTB.

Real-time target-specific detection of loop-mediated isothermal amplification for white spot syndrome virus using fluorescence energy transfer-based probes.

Aiming to establish a target amplicon-specific detection system for loop-mediated isothermal amplification (LAMP), the fluorescent resonance energy transfer (FRET) probe technology was applied to develop the FRET LAMP platform. This report describes the development of the first FRET LAMP assay targeting white spot syndrome virus (WSSV) of penaeid shrimp. A successful accelerated WSSV LAMP was assembled first in a conventional oven and confirmed by gel electrophoresis and dot-blot hybridization. Subsequently, two additional FRET probes designed to target one loop region within WSSV LAMP amplicons were added to the same LAMP reaction. The reactions were carried out in a LightCycler (Roche) and significant FRET signals were detected in real time. Optimization of the reaction using plasmid DNA shortened the time for the detection of 10(2) copies of the target DNA to less than 70min. Cross reactivity was absent with WSSV-free or infectious hypodermal and hematopoietic necrosis virus-infected Penaeus vannamei samples. The performance of this system was comparable with that of a nested PCR assay from 21 WSSV-infected shrimp. Specifically detecting target amplicons and requiring no post-amplification manipulation, the novel FRET LAMP assay should allow indisputable detection of pathogens with minimized risks of amplicon contamination.