Concept: Laboratory techniques
Background. Current malaria diagnostics, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. Molecular methods such as PCR are highly sensitive, but remain too complex for field deployment. A new commercial molecular assay based on loop-mediated isothermal amplification (LAMP) was assessed for field use.Methods. Malaria LAMP (Eiken Chemical Co., Ltd., Japan) was evaluated in 272 outpatients at a rural Ugandan clinic, and compared with expert microscopy, nested PCR (nPCR) and quantitative PCR (qPCR). Two technicians performed the assay after three days of training, using two alternative blood sample preparation methods and visual interpretation of results by fluorescence.Results. Compared with three-well nPCR, the sensitivity of both LAMP and single-well nPCR was 90%; microscopy sensitivity was 51%. For samples with P. falciparum qPCR titer≥2 parasites/µL, LAMP sensitivity was 97.8% (95% CI 93.7%-99.5%). Most false-negative LAMP results occurred in samples with parasitemia detectable by three-well nPCR but very low or undetectable by qPCR.Conclusions. Malaria LAMP in a remote Ugandan clinic achieved sensitivity similar to single-well nPCR in a UK reference laboratory. LAMP dramatically lowers the detection threshold achievable in endemic settings, providing a new tool for diagnosis, surveillance, and screening in elimination strategies.
Quantitative PCR assays are now the standard method for viral diagnostics. These assays must be specific, as well as sensitive, to detect the potentially low starting copy number of viral genomic material. We describe a new technique, polymerase chain displacement reaction (PCDR), which uses multiple nested primers in a rapid, capped, one-tube reaction that increases the sensitivity of normal quantitative PCR (qPCR) assays. Sensitivity was increased by approximately 10-fold in a proof-of-principle test on dengue virus sequence. In PCDR, when extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by utilizing a polymerase that has strand displacement activity. This allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Increased sensitivity in PCDR would be useful in nucleic acid detection for viral diagnostics.
, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of DNA extracted by each kit was performed using the specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (C ) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×10 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.
Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America, where sporadic outbreaks in cattle and pigs can cause clinical signs that are similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific differential diagnostic assays that are suitable for decision making in the field. RT-LAMP assays have been developed for vesicular diseases such as FMD and swine vesicular disease (SVD) but there is currently no RT-LAMP assay that can detect VS virus (VSV), nor are there any multiplex RT-LAMP assays which permit rapid discrimination between these ‘look-a-like’ diseases in situ. This study describes the development of a novel RT-LAMP assay for the detection of VSV focusing on the New Jersey (VSNJ) serotype, which has caused most of the recent VS cases in the Americas. This RT-LAMP assay was combined in a multiplex format combining molecular lateral-flow devices for the discrimination between FMD and VS. This assay was able to detect representative VSNJV and the limit of detection of the singleplex and multiplex VSNJV RT-LAMP assays were equivalent to laboratory based real-time RT-PCR assays. A similar multiplex RT-LAMP assay was developed to discriminate between FMDV and SVDV, showing that FMDV, SVDV and VSNJV could be reliably detected within epithelial suspensions without the need for prior RNA extraction, providing an approach that could be used as the basis for a rapid and low cost assay for differentiation of FMD from other vesicular diseases in the field.
The camel seminal plasma contains a diverse array of components including lipids, carbohydrates, peptides, ions and proteins. These are essential for maintaining normal physiology of spermatozoa and are secreted mainly from the prostrate, epidydimis and bulbo-urethral glands of reproductive system. The protein profiles of camel seminal plasma were resolved by two-dimensional gel electrophoresis (2D-PAGE). The majority of the protein was found in acidic regions below pI 7.0 and the 19 brightly stained proteins were identified by MALDI-TOF/MS analysis. On the basis of proteomic profiles, β-nerve growth factor (β-NGF) was purified by ion-exchange and gel filtration chromatography and identified by SDS-PAGE and MALDI-TOF/MS analysis. It was further confirmed by western blotting experiments using rabbit anti-β-NGF primary antibody.
Greenshell™ mussels (Perna canaliculus) were dry-stored at 6.44±0.54°C for 8 days during which time volatile organic compounds (VOCs) were monitored using SPME GC-MS. Thirty-four VOCs were identified in homogenised mussel meat and 29 in the mussel liquor (i.e. the seawater enclosed in the mantle cavity). Of the 34 VOCs identified 20 were reliably identified throughout the storage treatment and 9 were found to change in relative concentration in homogenised mussel meat. Dimethyl sulphide, 1-penten-3-ol, 1-hexen-3-ol and 1-octen-3-ol increased during storage, whereas pentanal, hexanal, heptanal, octanal and 3-undecen-2-one decreased. In the mussel liquor, dimethyl sulphide was undetectable pre-storage, becoming detectable after 2 days, and a large increase was noted after 6 days. SPME GC-MS was a useful tool for monitoring VOC profiles of Greenshell™ mussels and could aid in the development of technologies that monitor and improve product quality and consistency.
Bordetella pertussis causes an upper respiratory infection in infants, adolescents, and adults. Diagnosis of pertussis, a vaccine-preventable disease, can be difficult, but recent implementation of real-time PCR assays in laboratories has hastened the ability of clinicians to make an accurate diagnosis. In this paper we describe the method of nasopharyngeal specimen collection, extraction of DNA, and real-time PCR assays that will allow the detection and identification of Bordetella spp. in clinical specimens.
- Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society
- Published over 5 years ago
OBJECTIVE: The present study was performed to elucidate the possible role of SIRT1 signaling in joint inflammation in human articular chondrocytes. DESIGN: Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting were performed to detect gene products and proteins involved in tumor necrosis factor α (TNF-α)-induced inflammation and cartilage degradation in human primary chondrocytes. Matrix metalloproteinase (MMP)-2 and MMP-9 activity was evaluated by gelatin zymography. Overexpression and knockdown of SIRT1 were also performed to investigate whether SIRT1 is associated with the anti-inflammatory activity of resveratrol in chondrocytes. RESULTS: Resveratrol dose-dependently inhibited TNF-α-induced COX-2, MMP-1, MMP-3, MMP-13 and PGE(2) production in human chondrocytes. Moreover, MMP-2 and MMP-9 activity was increased by treatment with TNF-α; however, SIRT1 activation decreased the pro-inflammatory effects induced by TNF-α. In addition, treatment of SIRT1 activator and overexpression of SIRT1 inhibited the expression and activation of the main pro-inflammatory regulator NF-κB, which was increased by TNF-α. When SIRT1 was overexpressed in chondrocytes, the anti-inflammatory action of SIRT1 was similar to that exerted by resveratrol. CONCLUSIONS: SIRT1 activation deacetylates and inactivates NF-κB, and thereby, exerts an anti-inflammatory effect on chondrocytes, suggesting that SIRT1 activators could be explored as potential treatments for arthritis.
Abstract A solute aspirated into a prefilled tube of diluent undergoes a dilution effect known as dispersion. Traditionally the effects of dispersion have been considered a negative consequence of using liquid-filled fixed-tip liquid handlers. We present a novel device and technique that utilizes the effects of dispersion to the benefit of making dilutions. The device known as the Gradient Diluter extends the dilution range of practical serial dilutions to six orders of magnitude in final volumes as low as 10 μL. Presented are the device, dispersion methods, and validation tests using fluorescence detection of sulforhodamine and the high-performance liquid chromatography/ultraviolet detection of furosemide. In addition, a T-cell inhibition assay of a relevant downstream protein is used to demonstrate IC(50) curves made with the Gradient Diluter compare favorably with those generated by hand.
OBJECTIVE: MicroRNAs (miRNAs) are recognised as important regulators of a variety of fundamental biologic processes. Our study was undertaken to examine the role of MicroRNA-203 (miR-203) in modulating nitric oxide (NO) expression in female Sprague-Dawley rat mandibular condylar chondrocytes (MCCs) via targeting transient receptor potential vanilloid 4 (TRPV4) and to demonstrate the possible mechanism of NO inhibition by chondroprotective factor 17β-oestradiol (E2). METHODS: The expression of TRPV4 in mandibular condylar cartilage tissue and MCCs was detected by immunohistochemistry, immunofluorescence (IF), RT-PCR and Western blot, respectively. Primary SD rat MCCs were exposed to lipopolysaccharide (LPS), plus Ruthenium Red, 4α-phorbol 12,13-didecanoate (4αPDD), over-expressed miR-203 or E2 (10(-9) to 10(-6)M), the cellular supernatants were used for NO assay, miR-203 levels were measured by quantitative RT-PCR while TRPV4 expression changes were analysed by Western blot. The dual luciferase activity assay was performed to identify the target gene of miR-203. RESULTS: TRPV4 and miR-203 were stably expressed in MCCs. The MCCs' expression of NO evoked by LPS could be enhanced or depressed by Ruthenium Red or 4αPDD. The dual luciferase assay suggested that TRPV4 was the direct target gene of miR-203. Over-expression of miR-203 inhibited the expression of TRPV4 and increased NO expression in MCCs. E2 inhibited NO expression by inhibition of miR-203, which was concurrent with the up-regulation of TRPV4 expression level in MCCs. CONCLUSION: Our findings first suggested that miR-203 could up-regulate NO expression in female rat MCCs via targeting TRPV4. Moreover, the inhibition of NO by E2 might be at least in part through this mechanism.