scholarly journals Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification

2019 ◽  
Vol 2 (2) ◽  
pp. 36
Author(s):  
Lorena G. Caligiuri ◽  
Adolfo E. Sandoval ◽  
Jose C. Miranda ◽  
Felipe A. Pessoa ◽  
María S. Santini ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Internal Control ◽  
Large Scale ◽  
Pcr Amplification ◽  
Proteinase K ◽  
Sand Fly ◽  
Regulatory Function ◽  
Sand Flies ◽  
Lysis Buffer ◽  
Commercial Kits

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analyzing field-collected Lutzomyia spp. by polymerase chain reaction (PCR) and, for this reason, we evaluated various modifications on a previously published protocol, the most significant of which was a different lysis buffer that contained Ca2+ (buffer TESCa). This ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

scholarly journals Optimisation of DNA Extraction from Individual Sand Flies for PCR Amplification

2018 ◽  
Author(s):  
Lorena G. Caligiuri ◽  
Adolfo E. Sandoval ◽  
Jose C. Miranda ◽  
Felipe A. Pessoa ◽  
María S. Santini ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Internal Control ◽  
Large Scale ◽  
Pcr Amplification ◽  
Personal Communication ◽  
Proteinase K ◽  
Sand Fly ◽  
Regulatory Function ◽  
Sand Flies ◽  
Lysis Buffer

Numerous protocols have been published for extracting DNA from phlebotomines. Nevertheless, their small size is generally an issue in terms of yield, efficiency, and purity, for large-scale individual sand fly DNA extractions when using traditional methods. Even though this can be circumvented with commercial kits, these are generally cost-prohibitive for developing countries. We encountered these limitations when analysing parasite infection in Lutzomyia spp. by PCR [1] and, for this reason, we evaluated various modifications on a previously published protocol ([2] and Acardi personal communication). The most significant variation was the use of a different lysis buffer [3] to which added Ca2+ (buffer TESCa), because this ion protects proteinase K against autolysis, increases its thermal stability, and could have a regulatory function for its substrate-binding site [4]. Individual sand fly DNA extraction success was confirmed by amplification reactions using internal control primers that amplify a fragment of the cacophony gene [5,6]. To the best of our knowledge, this is the first time a lysis buffer containing Ca2+ has been reported for the extraction of DNA from sand flies.

scholarly journals A modified SDS-based DNA extraction method from raw soybean

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Yimiao Xia ◽  
Fusheng Chen ◽  
Yan Du ◽  
Chen Liu ◽  
Guanhao Bu ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Extraction Method ◽  
Pcr Amplification ◽  
Isoamyl Alcohol ◽  
Monitoring Methods ◽  
Detection Techniques ◽  
Dna Yield ◽  
Seed Flour ◽  
Dna Extraction Method ◽  
Lysis Buffer

Abstract Soybean is the most important genetically modified (GM) oilseed worldwide. Regulations relating to the approval of biotech soybean varieties and product labeling demand accurate and reliable detection techniques to screen for GM soya. High-quality extracted DNA is essential for DNA-based monitoring methods. Thus, four widely used protocols (SDS, CTAB, DP305, and DNeasy Plant Mini Kit) were compared in the present study to explore the most efficient DNA extraction method for raw soya matrix. The SDS-based method showed the highest applicability. Then crucial factors influencing DNA yield and purity, such as SDS lysis buffer component concentrations and organic compounds used to isolate DNA, were further investigated to improve the DNA obtained from raw soybean seeds, which accounts for the innovation of this work. As a result, lysis buffer (2% SDS (w/v), 150 mM NaCl, 50 mM Tris/HCl, 50 mM EDTA, pH 8.0) and organic reagents including chloroform/isoamyl alcohol (24:1, v/v) (C: I), isopropanol, and ethanol corresponding to the extraction and first and second precipitation procedures, respectively, were used in the optimized SDS method. The optimized method was verified by extracting approximately 2020–2444 ng DNA/mg soybean with A260/280 ratios of 1.862–1.954 from five biotech and non-biotech soybean varieties. Only 0.5 mg of soya was required to obtain enough DNA for PCR amplification using the optimized SDS-based method. These results indicate that the screening protocol in the present study achieves the highest suitability and efficiency for DNA isolation from raw soya seed flour.

Extracting DNA from submerged pine wood

Genome ◽  
2004 ◽  
Vol 47 (5) ◽  
pp. 994-997 ◽  
Author(s):  
M Megan Reynolds ◽  
Claire G Williams
Keyword(s):  
Dna Extraction ◽  
Dna Analysis ◽  
Pcr Amplification ◽  
Pinus Palustris ◽  
Proteinase K ◽  
High Molecular Weight Dna ◽  
Bisphosphate Carboxylase ◽  
Extraction Protocol ◽  
High Sequence Homology ◽  
Reaction Sequencing

A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser®. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.Key words: conifers, wood, polymerase chain reaction, sequencing.

scholarly journals Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections

2018 ◽  
Author(s):  
Tom Beneke ◽  
François Demay ◽  
Edward Hookway ◽  
Nicole Ashman ◽  
Heather Jeffery ◽  
...  
Keyword(s):  
Life Cycle ◽  
Large Scale ◽  
Gene Knockout ◽  
Sand Fly ◽  
Relative Fitness ◽  
Lutzomyia Longipalpis ◽  
Sand Flies ◽  
Label Free ◽  
Flagellar Proteins

AbstractThe protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite’s life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single mutant lacking the central pair protein PF16 showed that these paralysed promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.Author SummaryLeishmania are protozoan parasites, transmitted between mammals by the bite of phlebotomine sand flies. Promastigote forms in the sand fly have a long flagellum, which is motile and used for anchoring the parasites to prevent clearance with the digested blood meal remnants. To dissect flagellar functions and their importance in life cycle progression, we generated here a comprehensive list of >300 flagellar proteins and produced a CRISPR-Cas9 gene knockout library of 100 mutant Leishmania. We studied their behaviour in vitro before examining their fate in the sand fly Lutzomyia longipalpis. Measuring mutant swimming speeds showed that about half behaved differently compared to the wild type: a few swam faster, many slower and some were completely paralysed. We also found a group of uncoordinated swimmers. To test whether flagellar motility is required for parasite migration from the fly midgut to the foregut from where they reach the next host, we infected sand flies with a mixed mutant population. Each mutant carried a unique tag and tracking these tags up to nine days after infection showed that paralysed and uncoordinated Leishmania were rapidly lost from flies. These data indicate that directional swimming is important for successful colonisation of sand flies.

scholarly journals Development and Evaluation of PCR Assays for the Detection of Paenibacillus larvae in Honey Samples: Comparison with Isolation and Biochemical Characterization

2003 ◽  
Vol 69 (3) ◽  
pp. 1504-1510 ◽  
Author(s):  
Tamás Bakonyi ◽  
Irmgard Derakhshifar ◽  
Elvira Grabensteiner ◽  
Norbert Nowotny
Keyword(s):  
Dna Extraction ◽  
Large Scale ◽  
Biochemical Characterization ◽  
Direct Detection ◽  
Extraction Methods ◽  
Proteinase K ◽  
Paenibacillus Larvae ◽  
Lower Sensitivity ◽  
Pcr Assays ◽  
Dna Extraction Methods

ABSTRACT PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.

scholarly journals A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification.

1995 ◽  
Vol 4 (6) ◽  
pp. 368-370 ◽  
Author(s):  
D Goldenberger ◽  
I Perschil ◽  
M Ritzler ◽  
M Altwegg
Keyword(s):  
Dna Extraction ◽  
Extraction Procedure ◽  
Pcr Amplification ◽  
Proteinase K ◽  
Direct Pcr

Systemic Control of Cutaneous Leishmaniasis Sand-Fly Vectors: Fipronil-Treated Rodent Bait Is Effective in Reducing Phlebotomus papatasi (Diptera: Psychodidae) Female Emergence Rate From Rodent Burrows

2020 ◽  
Author(s):  
Ido Tsurim ◽  
Gideon Wasserberg ◽  
Gil Ben Natan ◽  
Zvika Abramsky
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Large Scale ◽  
Weak Link ◽  
Transmission Rate ◽  
Reservoir Host ◽  
Pathogen Transmission ◽  
Sand Fly ◽  
Sand Flies ◽  
Systemic Control ◽  
Emergence Rate

Abstract The strong dependency of some vectors on their host as a source of habitat can be viewed as a weak link in pathogen’s transmission cycles using the vertebrate host as a ‘Trojan horse’ to deliver insecticides directly to the vector-host point of contact (hereafter ‘systemic control’). This could, simultaneously, affect the survival of blood-feeding females and coprophagic larvae. Sand-flies, vectors of leishmaniasis worldwide, are often dependent on their bloodmeal host as a source of habitat and may therefore be good candidates for systemic control. In the present study, we field-tested this methodology by baiting Meriones crassus (Sundevall, 1842) (Rodentia:Muridea) with Fipronil-treated food pellets and evaluated its effect on reducing sand-fly emergence rate, in general, and of that of blood-fed females, in particular. We demonstrated 86% reduction in the abundance of female sand-flies that exit burrows of Fipronil-treated jirds, whereas male abundance was unaffected. Furthermore, whereas in control burrows 20% of the females were blood-fed, in treatment burrows no blood-fed females were detected. Sand-fly abundance outside the burrows was not affected by burrow treatment. This highlights the focal specificity of this method: affecting female sand-flies that feed on the reservoir host. This should result in the reduction of the pathogen transmission rate in the vicinity of the treated area by reducing the prevalence of leishmania-infected sand-fly females. These results hold promise for the potential of the systemic control approach in this system. Our next-step goal is to test this methodology at a large-scale cutaneous leishmaniasis control program.

scholarly journals Sand flies (Diptera: Psychodidae) in eight Balkan countries: historical review and region-wide entomological survey

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Vit Dvorak ◽  
Ozge Erisoz Kasap ◽  
Vladimir Ivovic ◽  
Ognyan Mikov ◽  
Jovana Stefanovska ◽  
...  
Keyword(s):  
Bosnia And Herzegovina ◽  
Large Scale ◽  
New Records ◽  
Historical Data ◽  
Historical Review ◽  
Sand Fly ◽  
Morphological Identification ◽  
Sand Flies ◽  
Entomological Survey ◽  
Balkan Countries

Abstract Background Sand flies (Diptera: Psychodidae) are medically important vectors of human and veterinary disease-causing agents. Among these, the genus Leishmania (Kinetoplastida: Trypanosomatidae), and phleboviruses are of utmost importance. Despite such significance, updated information about sand fly fauna is missing for Balkan countries where both sand flies and autochtonous leishmaniases are historically present and recently re-emerging. Therefore, a review of historical data on sand fly species composition and distribution in the region was followed by a large-scale entomological survey in eight Balkan countries to provide a recent update on local sand fly fauna. Methods The literature search involved the period 1910–2019. The entomological survey was conducted at 1189 sampling stations in eight countries (Bulgaria, Bosnia and Herzegovina, Croatia, Kosovo, Montenegro, North Macedonia, Serbia and Slovenia), covering 49 settlements and 358 sampling sites between June and October in the years 2014 and 2016, accumulating 130 sampling days. We performed a total of 1189 trapping nights at these stations using two types of traps (light and CO2 attraction traps) in each location. Sampling was performed with a minimal duration of 6 (Montenegro) and a maximal of 47 days (Serbia) between 0–1000 m.a.s.l. Collected sand flies were morphologically identified. Results In total, 8490 sand fly specimens were collected. Morphological identification showed presence of 14 species belonging to genera Phlebotomus and Sergentomyia. Historical data were critically reviewed and updated with our recent findings. Six species were identified in Bosnia and Herzegovina (2 new records), 5 in Montenegro (2 new records), 5 in Croatia (2 new records), 9 in Bulgaria (5 new records), 11 in North Macedonia (1 new record), 10 in Serbia (no new records), 9 in Kosovo (3 new records) and 4 in Slovenia (no new records). Conclusions This study presents results of the first integrated sand fly fauna survey of such scale for the Balkan region, providing first data on sand fly populations for four countries in the study area and presenting new species records for six countries and updated species lists for all surveyed countries. Our findings demonstrate presence of proven and suspected vectors of several Leishmania species.

scholarly journals A Rapid and Inexpensive Method of DNA Extraction from Palmyra Palm (Borassus flabellifer)

2019 ◽  
pp. 1-5
Author(s):  
Soni Kumari ◽  
Ruby Rani ◽  
Jitesh Kumar ◽  
Ravi Ranjan Kumar ◽  
Tushar Ranjan
Keyword(s):  
Dna Extraction ◽  
Dna Isolation ◽  
Pcr Amplification ◽  
Tween 20 ◽  
Inexpensive Method ◽  
Rapid Procedure ◽  
Dna Yield ◽  
Lysis Buffer ◽  
Yield And Purity ◽  
Different Levels

Aims: The study aims to highlight the simple optimisation, inexpensive and rapid procedure for DNA isolation from tough leaves (Palmyra palm) without compromising the yield and purity of DNA. Study Design: Leaf of palmyra palm (Borassus flabellifer) was used to conduct the experiment followed by laboratory analysis, DNA extraction and PCR amplification. Results and Discussion: The results showed that different buffers examined for the extraction of DNA provided significantly different levels of yield and purity. DNA isolated by lysis buffers C showed satisfactory amplifications in PCR. The fingerprint we obtained by using the DNA extracted by these buffers provided higher resolution than those using buffers. Conclusion: This study suggests that grinding of Palmyra palm leaves with sterile sand or cover slips and inclusion of SDS, Tween 20, and NaCl (1.4 M) in the lysis buffer without the costly use of liquid nitrogen, PVP and β mercaptoethanol, provides a DNA yield of sufficient purity, suitable for PCR amplification and subsequent use.

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