ResearchPad - dna-extraction https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[A tale of textiles: Genetic characterization of historical paper mulberry barkcloth from Oceania]]> https://www.researchpad.co/article/elastic_article_15748 Humans introduced paper mulberry (Broussonetia papyrifera) from Taiwan into the Pacific over 5000 years ago as a fiber source to make barkcloth textiles that were, and still are, important cultural artifacts throughout the Pacific. We have used B. papyrifera, a species closely associated to humans, as a proxy to understand the human settlement of the Pacific Islands. We report the first genetic analysis of paper mulberry textiles from historical and archaeological contexts (200 to 50 years before present) and compare our results with genetic data obtained from contemporary and herbarium paper mulberry samples. Following stringent ancient DNA protocols, we extracted DNA from 13 barkcloth textiles. We confirmed that the fiber source is paper mulberry in nine of the 13 textiles studied using the nuclear ITS-1 marker and by statistical estimates. We detected high genetic diversity in historical Pacific paper mulberry barkcloth with a set of ten microsatellites, showing new alleles and specific genetic patterns. These genetic signatures allow tracing connections to plants from the Asian homeland, Near and Remote Oceania, establishing links not observed previously (using the same genetic tools) in extant plants or herbaria samples. These results show that historic barkcloth textiles are cultural materials amenable to genetic analysis to reveal human history and that these artifacts may harbor evidence of greater genetic diversity in Pacific B. papyrifera in the past.

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<![CDATA[Democratizing water monitoring: Implementation of a community-based qPCR monitoring program for recreational water hazards]]> https://www.researchpad.co/article/elastic_article_14486 Recreational water monitoring can be challenging due to the highly variable nature of pathogens and indicator concentrations, the myriad of potential biological hazards to measure for, and numerous access points, both official and unofficial, that are used for recreation. The aim of this study was to develop, deploy, and assess the effectiveness of a quantitative polymerase chain reaction (qPCR) community-based monitoring (CBM) program for the assessment of bacterial and parasitic hazards in recreational water. This study developed methodologies for performing qPCR ‘in the field,’ then engaged with water management and monitoring groups and tested the method in a real-world implementation study to evaluate the accuracy of CBM using qPCR both quantitatively and qualitatively. This study found high reproducibility between qPCR results performed by non-expert field users and expert laboratory results, suggesting that qPCR as a methodology could be amenable to a CBM program.

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<![CDATA[Rediscovering an old foe: Optimised molecular methods for DNA extraction and sequencing applications for fungarium specimens of powdery mildew (Erysiphales)]]> https://www.researchpad.co/article/elastic_article_14476 The purpose of this study was to identify a reliable DNA extraction protocol to use on 25-year-old powdery mildew specimens from the reference collection VPRI in order to produce high quality sequences suitable to address taxonomic phylogenetic questions. We tested 13 extraction protocols and two library preparation kits and found the combination of the E.Z.N.A.® Forensic DNA kit for DNA extraction and the NuGen Ovation® Ultralow System library preparation kit was the most suitable for this purpose.

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<![CDATA[Quantitative real-time PCR as a promising tool for the detection and quantification of leaf-associated fungal species – A proof-of-concept using Alatospora pulchella]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc5cf

Traditional methods to identify aquatic hyphomycetes rely on the morphology of released conidia, which can lead to misidentifications or underestimates of species richness due to convergent morphological evolution and the presence of non-sporulating mycelia. Molecular methods allow fungal identification irrespective of the presence of conidia or their morphology. As a proof-of-concept, we established a quantitative real-time polymerase chain reaction (qPCR) assay to accurately quantify the amount of DNA as a proxy for the biomass of an aquatic hyphomycete species (Alatospora pulchella). Our study showed discrimination even among genetically closely-related species, with a high sensitivity and a reliable quantification down to 9.9 fg DNA (3 PCR forming units; LoD) and 155.0 fg DNA (47 PCR forming units; LoQ), respectively. The assay’s specificity was validated for environmental samples that harboured diverse microbial communities and likely contained PCR-inhibiting substances. This makes qPCR a promising tool to gain deeper insights into the ecological roles of aquatic hyphomycetes and other microorganisms.

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<![CDATA[Advanced approach to analyzing calcareous protists for present and past pelagic ecology: Comprehensive analysis of 3D-morphology, stable isotopes, and genes of planktic foraminifers]]> https://www.researchpad.co/article/5c8acce4d5eed0c484990244

Marine protists play an important role in oceanic ecosystems and biogeochemical cycles. However, the difficulties in culturing pelagic protists indicate that their ecology and behavior remain poorly understood; phylogeographic studies based on single-cell genetic analyses have often shown that they are highly divergent at the biological species level, with variable geographic distributions. This indicates that their ecology could be complex. On the other hand, the biomineral (calcareous) shells of planktic foraminifers are widely used in geochemical analyses to estimate marine paleoenvironmental characteristics (i.e., temperature), because the shell chemical composition reflects ambient seawater conditions. Among the pelagic protists, planktic foraminifers are ideal study candidates to develop a combined approach of genetic, morphological, and geochemical methods, thus reflecting environmental and ecological characteristics. The present study precisely tested whether the DNA extraction process physically and chemically affects the shells of the planktic foraminifer Globigerinoides ruber. We used a nondestructive method for analyzing physical changes (micro-focus X-ray computed tomography (MXCT) scanning) to compare specimens at the pre- and post-DNA extraction stages. Our results demonstrate that DNA extraction has no significant effect on shell density and thickness. We measured stable carbon and oxygen isotopes on the shell of each individual in a negative control or one of two DNA-extracted groups and detected no significant differences in isotopic values among the three groups. Moreover, we evaluated isotopic variations at the biological species level with regard to their ecological characteristics such as depth habitat, life stages, and symbionts. Thus, our examination of the physiochemical effects on biomineral shells through DNA extraction shows that morphological and isotopic analyses of foraminifers can be combined with genetic analysis. These analytical methods are applicable to other shell-forming protists and microorganisms. In this study, we developed a powerful analytical tool for use in ecological and environmental studies of modern and past oceans.

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<![CDATA[A survey on Mycobacterium ulcerans in Mosquitoes and March flies captured from endemic areas of Northern Queensland, Australia]]> https://www.researchpad.co/article/5c784fb8d5eed0c4840073ed

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU). This nontuberculous mycobacterial infection has been reported in 34 countries worldwide. In Australia, the majority of cases of BU have been recorded in coastal Victoria and the Mossman-Daintree areas of north Queensland. Mosquitoes have been postulated as a vector of M. ulcerans in Victoria, however the specific mode of transmission of this disease is still far from being well understood. In the current study, we trapped and analysed 16,900 (allocated to 845 pools) mosquitoes and 296 March flies from the endemic areas of north Queensland to examine for the presence of M. ulcerans DNA by polymerase chain reaction. Seven of 845 pools of mosquitoes were positive on screening using the IS2404 PCR target (maximum likelihood estimate 0.4/1,000). M. ulcerans DNA was detected from one pool of mosquitoes from which all three PCR targets: IS2404, IS2606 and the ketoreductase B domain of mycolactone polyketide synthase gene were detected. None of the March fly samples were positive for the presence of M. ulcerans DNA.

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<![CDATA[Prevalence of infection by the microsporidian Nosema spp. in native bumblebees (Bombus spp.) in northern Thailand]]> https://www.researchpad.co/article/5c8accecd5eed0c48499033b

Bumblebees (tribe Bombini, genus Bombus Latreille) play a pivotal role as pollinators in mountain regions for both native plants and for agricultural systems. In our survey of northern Thailand, four species of bumblebees (Bombus (Megabombus) montivagus Smith, B. (Alpigenobombus) breviceps Smith, B. (Orientalibombus) haemorrhoidalis Smith and B. (Melanobombus) eximius Smith), were present in 11 localities in 4 provinces (Chiang Mai, Mae Hong Son, Chiang Rai and Nan). We collected and screened 280 foraging worker bumblebees for microsporidia (Nosema spp.) and trypanosomes (Crithidia spp.). Our study is the first to demonstrate the parasite infection in bumblebees in northern Thailand. We found N. ceranae in B. montivagus (5.35%), B. haemorrhoidalis (4.76%), and B. breviceps (14.28%) and N. bombi in B. montivagus (14.28%), B. haemorrhoidalis (11.64%), and B. breviceps (28.257%).

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<![CDATA[How to integrate wet lab and bioinformatics procedures for wine DNA admixture analysis and compositional profiling: Case studies and perspectives]]> https://www.researchpad.co/article/5c6c759cd5eed0c4843cff14

The varietal authentication of wines is fundamental for assessing wine quality, and it is part of its compositional profiling. The availability of historical, cultural and chemical composition information is extremely important for quality evaluation. DNA-based techniques are a powerful tool for proving the varietal composition of a wine. SSR-amplification of genomic residual Vitis vinifera DNA, namely Wine DNA Fingerprinting (WDF) is able to produce strong, analytical evidence concerning the monovarietal nature of a wine, and for blended wines by generating the probability of the presence/absence of a certain variety, all in association with a dedicated bioinformatics elaboration of genotypes associated with possible varietal candidates. Together with WDF we could exploit Bioinformatics techniques, due to the number of grape genomes grown. In this paper, the use of WDF and the development of a bioinformatics tool for allelic data validation, retrieved from the amplification of 7 to 10 SSRs markers in the Vitis vinifera genome, are reported. The wines were chosen based on increasing complexity; from monovarietal, experimental ones, to commercial monovarietals, to blended commercial wines. The results demonstrate that WDF, after calculation of different distance matrices and Neighbor-Joining input data, followed by Principal Component Analysis (PCA) can effectively describe the varietal nature of wines. In the unknown blended wines the WDF profiles were compared to possible varietal candidates (Merlot, Pinot Noir, Cabernet Sauvignon and Zinfandel), and the output graphs show the most probable varieties used in the blend as closeness to the tested wine. This pioneering work should be meant as to favor in perspective the multidisciplinary building-up of on-line databanks and bioinformatics toolkits on wine. The paper concludes with a discussion on an integrated decision support system based on bioinformatics, chemistry and cultural data to assess wine quality.

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<![CDATA[Viable Neisseria meningitidis is commonly present in saliva in healthy young adults: Non-invasive sampling and enhanced sensitivity of detection in a follow-up carriage study in Portuguese students]]> https://www.researchpad.co/article/5c6b2652d5eed0c48428980e

Introduction and aims

Improved sensitivity and efficiency of detection and quantification of carriage of Neisseria meningitidis (Nm) in young people is important for evaluation of the impact of vaccines upon transmission and associated population-wide effects. Saliva collection is quick, non-invasive and facilitates frequent sampling, but has been reported to yield low sensitivity by culture. We re-evaluated this approach in a follow-up cross sectional study using direct and culture-amplified PCR.

Material/Methods

In April 2016 we collected paired oropharyngeal swabs (OPS) and saliva samples from 1005 healthy students in Portugal into STGG broth and stored them at -80°C until DNA extraction and batched qPCR analysis. Samples were also cultured on GC agar plates for 72h and PCR done on DNA extracts from overall growth. Nm isolates were also sought from a selection of 50 samples. qPCR amplification targets were superoxide dismutase sodC and capsular locus/genogroup-specific genes (B, C, W, X and Y) and, for cultured isolates only, porA. Cycle threshold values of ≤36 were considered positive.

Results

556 tests (460 samples, 363 subjects, 36.1%) were positive for Nm (sodC) and 65 (45, 36, 3.6%) for MenB. More salivas were positive by direct sodC qPCR (211, 21.0%) than OPS (126, 12.5%) but fewer were positive by culture-amplified qPCR (94 vs. 125). For both sample types, many that were negative on direct qPCR came positive on culture-amplification and Nm was consistently isolated from salivas in which culture amplified the PCR signal. Using both methods on both samples yielded 36.1% Nm and 5.5% encapsulated Nm carriage rates while direct qPCR on OPS alone detected 12.5% and 2.2%.

Conclusions

Detectable MenB carriage rates (2.9%) were lower than 4 years earlier (6.8%) in this population (p = 0.0003). Viable meningococci were often present in saliva. Although evidence of encapsulated Nm was less frequent in saliva than OPS, collection is more acceptable to subjects allowing more frequent sampling. Use of culture-amplification increases detection sensitivity in both sample types, especially when combined with direct PCR. Combining these samples and/or methodologies could greatly enhance the power of carriage studies to detect the impact of vaccines upon carriage and transmission.

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<![CDATA[Quality and quantity of dromedary camel DNA sampled from whole-blood, saliva, and tail-hair]]> https://www.researchpad.co/article/5c5ca302d5eed0c48441efd7

Camels are livestock with unique adaptations to hot-arid regions. To effectively study camel traits, a biobank of camel DNA specimens with associated biological information is needed. We examined whole-blood, saliva (buccal swabs), and tail-hair follicle samples to determine which is the best source for establishing a DNA biobank. We inspected five amounts of each of whole-blood, buccal swabs, and tail-hair follicles in nine camels, both qualitatively via gel electrophoresis and quantitatively using a NanoDrop spectrophotometer. We also tested the effects of long term-storage on the quality and quantity of DNA, and measured the rate of degradation, by analyzing three buccal swab samples and 30 tail-hair follicles over a period of nine months. Good quality DNA, in the form of visible large size DNA bands, was extracted from all three sources, for all five amounts. The five volumes of whole-blood samples (20–100μl) provided ~0.4–3.6 μg, the five quantities of buccal swabs (1–5) produced ~0.1–12 μg, while the five amounts of tail-hair follicles (10–50) resulted in ~0.7–25 μg. No differences in the rate of degradation of buccal swab and tail-hair follicle DNA were detected, but there was clearly greater deterioration in the quality of DNA extracted from buccal swabs when compared to tail-hair follicles. We recommend using tail-hair samples for camel DNA biobanking, because it resulted in both an adequate quality and quantity of DNA, along with its ease of collection, transportation, and storage. Compared to its success in studies of other domesticated animals, we anticipate that using ~50 tail-hair follicles will provide sufficient DNA for sequencing or SNP genotyping.

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<![CDATA[Prevalence of high-risk human papilloma virus in women with high-grade squamous cell intraepithelial lesions in Botswana using Abbott RealTime HPV assay]]> https://www.researchpad.co/article/5c5b52a6d5eed0c4842bcd6b

Background

High-risk human papillomavirus (HR-HPV) has been demonstrated to be the necessary cause of cervical carcinoma. High-risk HPV detection has a prognostic significance for the women who are at increased risk of disease progression. HPV genotyping in cervical cancer precursor lesions is crucial for prevention and management of cervical cancer. This study was designed to investigate the distribution of HR-HPV genotypes among a group of patients with high-grade squamous intraepithelial lesions and higher, of the cervix, in Botswana.

Materials and methods

185-archived residual formalin-fixed paraffin-embedded cervical biopsies collected between the years, 2006 and 2008 were studied. These tissues were diagnosed with HSIL (n = 146) and squamous cell carcinoma (n = 39). DNA was extracted using the Abbott m2000 analyser (Abbott Laboratories, Illinois) using reagents provided by the manufacturer. HPV genotyping was done using the Abbott RealTime HR-HPV PCR, which qualitatively detects 14 HR-HPV (reported as HPV 16, 18 & Other HR-HPV).

Results

DNA was successfully extracted from 162/185 (87.6%) tissues as indicated by a positive β-globin test. 132/162 (82%) tested positive for HR-HPV The HPV 16 prevalence was 50% (66/132), HPV 18 at 15.2% (20/132) and other Group 1 HR-HPV plus HPV 66 and 68 had a prevalence of 56.1% (74/132). Other HR-HPV types were common in HSIL than in carcinoma, while HPV 16 was more prevalent in carcinomas than other HR-HPV genotypes.

Conclusion

In this study, HPV 16 and other HR-HPV genotypes were commonly associated with HSIL but HPV 18 was uncommon among Botswana women. Our data highlights the need for multivalent HPV vaccines with cross coverage for other high risk HPV other than HPV 16 and 18.

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<![CDATA[Improving cost-efficiency of faecal genotyping: New tools for elephant species]]> https://www.researchpad.co/article/5c5b527bd5eed0c4842bc95f

Despite the critical need for non-invasive tools to improve monitoring of wildlife populations, especially for endangered and elusive species, faecal genetic sampling has not been adopted as regular practice, largely because of the associated technical challenges and cost. Substantial work needs to be undertaken to refine sample collection and preparation methods in order to improve sample set quality and provide cost-efficient tools that can effectively support wildlife management. In this study, we collected an extensive set of forest elephant (Loxodonta cyclotis) faecal samples throughout Gabon, Central Africa, and prepared them for genotyping using 107 single-nucleotide polymorphism assays. We developed a new quantitative polymerase chain reaction (PCR) assay targeting a 130-bp nuclear DNA fragment and demonstrated its suitability for degraded samples in all three elephant species. Using this assay to compare the efficacy of two sampling methods for faecal DNA recovery, we found that sampling the whole surface of a dung pile with a swab stored in a small tube of lysis buffer was a convenient method producing high extraction success and DNA yield. We modelled the influence of faecal quality and storage time on DNA concentration in order to provide recommendations for optimized collection and storage. The maximum storage time to ensure 75% success was two months for samples collected within 24 hours after defecation and extended to four months for samples collected within one hour. Lastly, the real-time quantitative PCR assay allowed us to predict genotyping success and pre-screen DNA samples, thus further increasing the cost-efficiency of our approach. We recommend combining the validation of an efficient sampling method, the build of in-country DNA extraction capacity for reduced storage time and the development of species-specific quantitative PCR assays in order to increase the cost-efficiency of routine non-invasive DNA analyses and expand the use of next-generation markers to non-invasive samples.

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<![CDATA[Metabarcoding targeting the EF1 alpha region to assess Fusarium diversity on cereals]]> https://www.researchpad.co/article/5c42436cd5eed0c4845e0155

Fusarium head blight (FHB) is a major cereal disease caused by a complex of Fusarium species. These species vary in importance depending on climatic conditions, agronomic factors or host genotype. In addition, Fusarium species can release toxic secondary metabolites. These mycotoxins constitute a significant food safety concern as they have health implications in both humans and animals. The Fusarium species involved in FHB differ in their pathogenicity, ability to produce mycotoxins, and fungicide sensitivity. Accurate and exhaustive identification of Fusarium species in planta is therefore of great importance. In this study, using a new set of primers targeting the EF1α gene, the diversity of Fusarium species on cereals was evaluated using Illumina high-throughput sequencing. The PCR amplification parameters and bioinformatic pipeline were optimized with mock and artificially infected grain communities and further tested on 65 field samples. Fusarium species were retrieved from mock communities and good reproducibility between different runs or PCR cycle numbers was be observed. The method enabled the detection of as few as one single Fusarium-infected grain in 10,000. Up to 17 different Fusarium species were detected in field samples of barley, durum and soft wheat harvested in France. This new set of primers enables the assessment of Fusarium diversity by high-throughput sequencing on cereal samples. It provides a more exhaustive picture of the Fusarium community than the currently used techniques based on isolation or species-specific PCR detection. This new experimental approach may be used to show changes in the composition of the Fusarium complex or to detect the emergence of new Fusarium species as far as the EF1α sequence of these species show a sufficient amount of polymorphism in the portion of sequence analyzed. Information on the distribution and prevalence of the different Fusarium species in a given geographical area, and in response to various environmental factors, is of great interest for managing the disease and predicting mycotoxin contamination risks.

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<![CDATA[Improved DNA extraction technique from clot for the diagnosis of Chagas disease]]> https://www.researchpad.co/article/5c424367d5eed0c4845e00a2

Background

The detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. We have previously demonstrated that PCR using clot samples yields greater sensitivity than either buffy coat or whole blood samples. However, phenol-chloroform DNA extraction from clot samples is difficult and toxic. The objective of the present study was to improve and develop a more sensitive method to recover parasite DNA from clot samples for the diagnosis of Chagas disease.

Methodology/Principal findings

A total of 265 match pair samples of whole blood–guanidine (GEB) and clot samples were analyzed; 150 were from Chagas seropositive subjects. DNA was extracted from both whole blood-guanidine samples, using a previously standardized methodology, and from clot samples, using a newly developed methodology based on a combination of the FastPrep technique and the standard method for GEB extraction. A qPCR targeting the nuclear satellite sequences was used to compare the sample source and the extraction method. Of the 150 samples from Chagas positive individuals by serology, 47 samples tested positive by qPCR with DNA extracted by both GEB and clot, but an additional 13 samples tested positive only in DNA extracted from clot. No serology-negative samples resulted positive when tested by qPCR.

Conclusions

The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease.

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<![CDATA[Rapid and sensitive detection of Chlamydia trachomatis sexually transmitted infections in resource-constrained settings in Thailand at the point-of-care]]> https://www.researchpad.co/article/5c254548d5eed0c48442c426 ]]> <![CDATA[Development of a PCR algorithm to detect and characterize Neisseria meningitidis carriage isolates in the African meningitis belt]]> https://www.researchpad.co/article/5c117bced5eed0c48469a800

Improved methods for the detection and characterization of carried Neisseria meningitidis isolates are needed. We evaluated a multiplex PCR algorithm for the detection of a variety of carriage strains in the meningitis belt. To further improve the sensitivity and specificity of the existing PCR assays, primers for gel-based PCR assays (sodC, H, Z) and primers/probe for real-time quantitative PCR (qPCR) assays (porA, cnl, sodC, H, E, Z) were modified or created using Primer Express software. Optimized multiplex PCR assays were tested on 247 well-characterised carriage isolates from six countries of the African meningitis belt. The PCR algorithm developed enabled the detection of N. meningitidis species using gel-based and real-time multiplex PCR targeting porA, sodC, cnl and characterization of capsule genes through sequential multiplex PCR assays for genogroups (A, W, X, then B, C, Y and finally H, E and Z). Targeting both porA and sodC genes together allowed the detection of meningococci with a sensitivity of 96% and 89% and a specificity of 78% and 67%, for qPCR and gel-based PCR respectively. The sensitivity and specificity ranges for capsular genogrouping of N. meningitidis are 67% - 100% and 98%-100% respectively for gel-based PCR and 90%-100% and 99%-100% for qPCR. We developed a PCR algorithm that allows simple, rapid and systematic detection and characterisation of most major and minor N. meningitidis capsular groups, including uncommon capsular groups (H, E, Z).

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<![CDATA[A novel approach to stabilize fetal cell-free DNA fraction in maternal blood samples for extended period of time]]> https://www.researchpad.co/article/5c12cf27d5eed0c484914028

This study was undertaken to evaluate a novel method for stabilizing and preserving the original proportion of cell-free fetal DNA (cffDNA) in maternal blood for extended periods of time without using crosslinking agents, such as formaldehyde, which compromise DNA integrity and extraction efficiency. Blood was drawn from pregnant donors into K3EDTA and Blood Exo DNA ProTeck® (ProTeck) tubes. Blood drawn into both tubes were aliquoted and stored at three different temperatures. At indicated times sample aliquots were processed for cell-free DNA (cfDNA) extraction. Plasma cfDNA and cffDNA quantified by droplet digital PCR (ddPCR) assay which amplify RASSF1A gene promoter region. ProTeck reagent is formaldehyde free and inhibits blood cell metabolism in blood samples during storage. Cell-free DNA concentration increased over time in blood plasma stored in K3EDTA tubes at 4, 22 and 30°C. Blood stored in ProTeck tubes, cfDNA concentration was stable at 4, 22 and 30°C for 21, 28 and 7 days, respectively. In K3EDTA tubes cffDNA proportion decreases steadily over time whereas in ProTeck tubes cffDNA proportion remained stable. This novel technology stabilizes cffDNA proportion in maternal blood samples at 4, 22 and 30°C for 21, 28 and 7 days, respectively.

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<![CDATA[Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL)]]> https://www.researchpad.co/article/5bf71f9ad5eed0c484dcb98e

Background

The World Health Organization has targeted elimination of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) by 2020. Despite distinctive decline seen in the number of VL cases in ISC, there is still a quest for development of a diagnostic test which has the utility for detection of active infection and relapse cases and as a test of cure. The present study validated the sensitivity and specificity of SYBR Green I based closed tube LAMP assay reported by us for diagnosis of VL.

Methodology

The validation study was carried out at two endemic sites in India, located at Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna and Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi. Standard operating protocols were provided at the two sites for applying LAMP assay on confirmed VL cases. The diagnostic accuracy of LAMP assay was evaluated by Receiver operator curve (ROC) analysis. Furthermore, a simplified LAMP assay based on direct blood lysis, DBL-LAMP, was developed and verified for its diagnostic accuracy.

Principal findings

A total of 267 eligible participants were included in the study which comprised of 179 VL cases and 88 controls. Sensitivity and specificity of the LAMP assay were 98.32% (95% C.I– 95.2–99.7%) and 96.59% (95% C.I.-90.4–99.3%), respectively. ROC curve analysis depicted no significant difference between area under curve (AUCROC) for LAMP assay and rK39 RDT, indicative of LAMP as an excellent diagnostic test. DBL-LAMP assay, performed on 67 VL and 100 control samples, yielded a sensitivity of 93.05% (95% C.I- 84.75–97%) and specificity of 100% (95% C.I.- 96.30–100%).

Conclusions/Significance

The validated closed tube LAMP for diagnosis of VL will provide impetus to the ongoing VL elimination programme in ISC. The assay based on direct blood lysis promotes its scope for application in field settings by further reducing time and cost.

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<![CDATA[Backpack PCR: A point-of-collection diagnostic platform for the rapid detection of Brugia parasites in mosquitoes]]> https://www.researchpad.co/article/5bff05b5d5eed0c484a339d2

Background

Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA detection assay. While the research reported here serves as a proof-of-concept for the backpack PCR methodology for the detection of filarial parasites in mosquitoes, the platform should be easily adaptable to the detection of W. bancrofti and other mosquito-transmitted pathogens.

Methodology/Principal findings

Through comparisons with standard silica column-based DNA extraction techniques, we evaluated the performance of a rapid, NaOH-based methodology for the extraction of total DNA from pools of parasite-spiked vector mosquitoes. We also compared our novel test strip-based detection assay to real-time PCR and conventional PCR coupled with gel electrophoresis, and demonstrated that this method provides sensitive and genus-specific detection of parasite DNA from extracted mosquito pools. Finally, by comparing laboratory-based thermal cycling with a field-friendly miniaturized PCR approach, we have demonstrated the potential for the point-of-collection-based use of this entire diagnostic platform that is compact enough to fit into a small backpack.

Conclusions/Significance

Because this point-of-collection diagnostic platform eliminates reliance on expensive and bulky instrumentation without compromising sensitivity or specificity of detection, it provides an alternative to cost-prohibitive column-dependent DNA extractions that are typically coupled to detection methodologies requiring advanced laboratory infrastructure. In doing so, this field-ready system should increase the feasibility of molecular xenomonitoring within B. malayi-endemic locations. Of greater importance, this backpack PCR system also provides the proof-of-concept framework for the development of a parallel assay for the detection of W. bancrofti.

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<![CDATA[Comparison of the upper and lower airway microbiota in children with chronic lung diseases]]> https://www.researchpad.co/article/5b6da1ac463d7e4dccc5fae9

Rationale

The lower airway microbiota is important in normal immunological development and chronic lung diseases (CLDs). Young children cannot expectorate and because of the uncertainty whether upper airway samples reflect the lower airway microbiota, there have been few longitudinal paediatric studies to date.

Objectives

To assess whether throat swabs (TS) and cough swabs (CS) are representative of the lower airway microbiota.

Methods

TS, CS, bronchoalveolar lavage and bronchial brushings were prospectively collected from 49 children undergoing fibreoptic bronchoscopy for CLDs. Bacterial DNA was extracted and the 16S rRNA gene V4 region sequenced using the Illumina MiSeq.

Results

5.97 million high quality reads were obtained from 168 samples (47 TS, 37 CS, 42 BALF and 42 bronchial brushings). CS sequenced poorly. At a community level, no difference in alpha diversity (richness, evenness or Shannon Diversity Index) was seen between lower airway samples and TS (P > 0.05). Less than 6.31% of beta diversity variation related to sampling method for TS (P = 0.001). Variation between pathologies and individual patients was greater (20%, 54% respectively P ≤ 0.001) than between TS and lower airway samples. There was strong correlation in the relative abundance of genera between samples (r = 0.78, P < 0.001). Similarity between upper and lower airway samples was observed to be less for individuals where one sample type was dominated by a single organism.

Conclusions

At the community structure level, TS correlate with lower airway samples and distinguish between different CLDs. TS may be a useful sample for the study of the differences in longitudinal changes in the respiratory microbiota between different CLDs. Differences are too great however for TS to be used for clinical decision making.

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