SciCombinator

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Concept: Laboratory techniques

179

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.

Concepts: Infectious disease, Polymerase chain reaction, Molecular biology, Malaria, Plasmodium falciparum, Type I and type II errors, Real-time polymerase chain reaction, Laboratory techniques

167

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.

Concepts: DNA, Polymerase chain reaction, Molecular biology, Real-time polymerase chain reaction, DNA replication, DNA polymerase, Laboratory techniques, Primer

166

, 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.

Concepts: DNA, Polymerase chain reaction, Molecular biology, Laboratory techniques, Burkholderia mallei, Burkholderia, Burkholderia pseudomallei, Melioidosis

158

We have developed and evaluated a real-time reverse transcription PCR (RT-PCR) assay for detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak associated with severe respiratory illness in the United States. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, Missouri. This sequence, along with other GenBank® sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP-1 gene with an analytic limit of detection of 4 copies per reaction, and was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested, and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses.

Concepts: DNA, Polymerase chain reaction, Molecular biology, Virus, Real-time polymerase chain reaction, Reverse transcriptase, Reverse transcription polymerase chain reaction, Laboratory techniques

146

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.

Concepts: Assay, Aphthovirus, Foot-and-mouth disease, Laboratory techniques, South America, Animal virology, Americas, Swine vesicular disease

43

During measles outbreaks, it is important to be able to rapidly distinguish between measles cases and vaccine reactions to avoid unnecessary outbreak response measures such as case isolation and contact investigations. We have developed a real-time RT-PCR method specific for genotype A measles virus (MeVA RT-qPCR), that can identify measles vaccine strains rapidly, with high throughput, and without the need for sequencing to determine the genotype. We have evaluated the method independently in three measles reference laboratories using two platforms, the Roche Lightcycler® 480 and the Applied Biosystems™ (ABI) 7500 Real-Time PCR System. In comparison to the standard real time RT-PCR method, the MeVA RT-qPCR showed 99.5% specificity for genotype A and 94% sensitivity for both platforms. The new assay was able to detect RNA from five currently used vaccine strains, AIK-C, CAM-70, Edmonston-Zagreb, Moraten, and Shanghai-191. The MeVA RT qPCR assay has been used successfully for measles surveillance in reference laboratories and it could be readily deployed to national and subnational laboratories on a wide scale.

Concepts: DNA, Polymerase chain reaction, Molecular biology, Vaccine, Real-time polymerase chain reaction, Reverse transcription polymerase chain reaction, Laboratory techniques, Measles

28

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.

Concepts: Protein, Molecular biology, Western blot, Gel electrophoresis, Electrophoresis, Laboratory techniques, Two-dimensional gel electrophoresis, Isoelectric focusing

28

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.

Concepts: Mass spectrometry, Laboratory techniques, Solid phase microextraction, Volatile organic compound, Organic compounds, Mussel, Volatile, New Zealand green-lipped mussel

28

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.

Concepts: Infectious disease, Polymerase chain reaction, Molecular biology, Biology, Bordetella pertussis, Laboratory techniques, Bordetella parapertussis

28

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.

Concepts: DNA, Gene expression, Polymerase chain reaction, Molecular biology, Histone deacetylase, Real-time polymerase chain reaction, Tumor necrosis factor-alpha, Laboratory techniques