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Surna Inc. (SRNA )

Strain-Profile User-Generated

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27.06.2018

Content:

  • Strain-Profile User-Generated
  • Jack Herer Strain (2019 Review)
  • Amnesia Haze Strain Review: Aroma, Flavor, and Appearance
  • SeedFinders PlantProfile-Generator enables you to write a standardized Strain- Profile including information about growing, smoking and effects of a. Oct 7, This cannabinoid profile makes the strain ideal for medical and recreational It is known to help combat pain and will cause users to develop quite the appetite. The head high generated by smoking White Widow flowers is. Feb 5, The smell of marijuana (Cannabis sativa L.) is of interest to users, growers, . A strain's aroma profile can be represented as frequency counts across in Fig 4, a composite odor profile was created for each of them (Fig 5).

    Strain-Profile User-Generated

    This is especially the case for bacteria exhibiting high levels of antibiotic resistance or virulence, and those involved in nosocomial or pandemic infections. Strain typing also has applications in studying bacterial population dynamics. Over the last two decades, molecular methods have progressively replaced phenotypic assays to type bacterial strains. In this article, we review the current bacterial genotyping methods and classify them into three main categories: We described and compared the applications of genotyping methods to the study of bacterial strain diversity.

    We also discussed the selection of appropriate genotyping methods and the challenges of bacterial strain typing, described the current trends of genotyping methods, and investigated the progresses allowed by the availability of genomic sequences.

    Genetic diversity can ultimately explain most phenotypic variability in bacteria, such as geographic distribution, host specificity, pathogenicity, antibiotic resistance, and virulence.

    As bacterial strains pose ever greater challenges to human health, including increased virulence and transmissibility, resistance to multiple antibiotics, expanding host spectra, and the possibility of genetic manipulation for bioterrorism, identifying bacteria at the strain level is increasingly important in modern microbiology Fournier et al. The intraspecies diversity of bacteria mainly results from three genetic events: The frequency of these three events makes the investigation of intraspecies diversity quite complicated Fraser-Liggett, Although whole-genome sequencing, followed by genome comparison, is in theory an ideal way to elucidate the genetic variability within a bacterial species, it remains expensive and labor intensive Field et al.

    Bacterial strain typing, characterizing a number of strains in detail and ascertaining whether they are derived from a single parental organism, is a way to identify bacteria at the strain level and to uncover the genetic diversity underlying important phenotypic characteristics. Theoretically, there are two distinct typing systems: Bacterial phenotypes, determined by the morphology of colonies on various culture media, biochemical tests, serology, killer toxin susceptibility, pathogenicity, and antibiotic susceptibility, are not variable enough for discriminating between closely related strains.

    In the genomic era, the scientific basis for the identification and subtyping of microorganisms has shifted to genetic methods. More recently, genotyping, which refers to the discrimination of bacterial strains based on their genetic content, has become widely used for bacterial strain typing due to its high resolution.

    The genetic profile of a given strain generated by a specific genotyping method can be as unique as a fingerprint. Thus, genotyping is also referred to as DNA fingerprinting. Current bacterial strain typing methods may be classified into three main categories: DNA sequencing-based genotyping methods generate the original sequence of nucleotides and discriminate among bacterial strains directly from polymorphisms in their DNA.

    In this technique, bacterial strains are discriminated by analyzing the hybridization of their DNA to probes of known sequences.

    With the exception of genome sequencing, the discriminatory ability of genotyping methods is species dependent. REs precisely recognize and cut target DNA at a defined sequence, which makes enzymatic restriction an effective tool Blakesley, In silico searches of completed bacterial genome sequences to detect recognition sites of rare-cutting REs proved to be an effective way to optimize the discriminatory power and reduce the cost of PFGE in the case of typing Bordetella pertussis Lee et al.

    A website dedicated to in silico RE analysis of complete bacterial genomes was developed by Bikandi and colleagues. Their strategy allows the calculation of the theoretical number and size of restriction fragments generated by different REs and provides a graphically detailed restriction profile when the number of fragments is 50 or fewer Bikandi et al.

    Nevertheless, PFGE results should be validated by comparison with epidemiological traits and other genotyping results. PFGE is now widely used in epidemiology, microbiology, and evolutionary biology.

    As proposed by Tenover and colleagues, bacterial isolates yielding the same PFGE profile are considered as belonging to the same strain. However, although widely used, PFGE has several limitations. This method is time and labor consuming and lacks reproducibility and interlaboratory comparability. In addition, it requires high-quality DNA, is poorly applicable to human or environmental samples, and may lack resolution power to distinguish bands of nearly identical size Davis et al.

    Other drawbacks include the risk of laboratory-acquired infection due to prolonged handling of bacterial strains before treating with proteases and REs, and many other factors such as concentration of DNA in the agarose plugs, amount of agarose in the gel, electrophoresis voltage, gel temperature, and buffer strength, which may also influence patterns Chung et al.

    As its name implies, RFLP measures the size of restriction fragments separated by conventional agarose gel electrophoresis. Digestion of genomic DNA with frequently cutting REs may produce hundreds of short restriction fragments, making clear separation of fragments difficult using agarose gel electrophoresis Table 1. However, RFLP analysis can be greatly simplified by subjecting the partial restriction fragments to Southern blotting with labeled probes Southern, In bacteriology, if two strains differ in the distance between cleavage sites for a particular RE, the length of the restriction fragments is different between the strains.

    The similarity of the generated patterns of restriction fragments can be used to differentiate strains and to analyze the genetic relatedness Busse et al. Bacterial rRNA operons comprise a family of highly conserved genes, each of which is flanked by more variable DNA regions.

    An advantage of ribotyping is that it enables analysis without prior knowledge of genomic DNA sequence, because rRNA operons are universal. In addition, the results of ribotyping are easier to interpret because fewer fragments are produced Bingen et al.

    This system provides highly reproducible and standardized ribotyping data. Ribobank is a ribotyping database comprising numerous bacterial ribotypes generated by Qualicon's RiboPrinter approach Table 2. Because it does not require expensive equipment, RFLP analysis is cost effective except for automatic ribotyping that requires a relatively expensive kit.

    In addition, it is time and labor consuming, especially when coupled with Southern blotting, and involves detection systems that use either radioisotopes or complex biochemistry.

    If a mutation has occurred in the template DNA at a site that was previously complementary to the primer, a PCR product may not be produced, and a different pattern of amplified DNA fragments is observed on the gel. Based on the observation that the number of fragments generated by AP-PCR correlates with the number of arbitrary primer annealing sites in the target genome sequence, Li et al. Some arbitrary primers, namely the universal rice primers extracted from a repetitive sequence in the rice genome Kang et al.

    It has widely been used to study the genetic variability of many bacterial species, including important human pathogens Power, ; Jin et al. However, the reproducibility of this method remains a challenge, which hinders comparison of AP-PCR patterns between and within laboratories Power, ; Tyler et al.

    This approach may prove useful for standardizing and optimizing this genotyping method Hyytia et al. A series of naturally occurring repetitive DNA sequences are dispersed in multiple copies throughout bacterial genomes Gilson et al. Although the functions of these interspersed repetitive DNA elements remain unknown, their presence is useful for DNA fingerprinting of bacteria.

    Three families of repetitive sequences: Detection of DNA fragments is obtained by agarose gel electrophoresis or capillary electrophoresis. The optimization of electrophoresis conditions electrokinetic injection and applied voltage can dramatically increase the resolution of amplified DNA fragments up to bp Sciacchitano, Because of the low cost of the materials and the rapidity, ease of use, and low labor intensity, REP-PCR may be a valuable method for bacterial strain typing.

    REP-PCR has been shown to be beneficial for typing eukaryotic and prokaryotic organisms, including bacteria of epidemiological significance, i. The online diversilab software not only provides standardized comparisons among isolates almost instantaneously, but also generates user-friendly customized reports and provides a user-specific data storage and retrieval system Healy et al.

    In bacterial genomes, VNTR loci were found in noncoding regions as well as in genes. These VNTR can be an important source of genetic polymorphism for strain typing because of their rapid evolution van et al. The number of tandem repeats per locus may vary dramatically between strains within a given species. For each VNTR locus, the number of repeats can be determined by PCR amplification using primers complementary to the well-conserved sequences flanking the tandem repeats.

    The fragment size varies with regard to the size and number of repeat units, and the banding patterns can be analyzed using a software to reveal the genotype and infer the phylogenetic relationships Keim et al.

    By fluorescently labeling PCR amplicons with different dye colors and separating them by capillary electrophoresis in an automated sequencer, MLVA can be performed in a single PCR tube multiplex PCR , which dramatically improves its efficiency Lindstedt, ; Lista et al.

    Tandem repeat-finding programs, such as tandem repeat finder Benson, , the Tandem Repeat database, and the Microsatellite Repeats database provide a series of bioinformatic tools for searching bacterial genomes for potential VNTR loci Table 2.

    Since its first use to type bacteria in , MLVA has proven to be a high-resolution method for discrimination of many bacteria. It is now regarded as a reference typing method for many bacterial species, such as Francisella tularensis Farlow et al. MLVA has also been applied to other important human pathogens such as methicillin-resistant Staphylococcus aureus strains Tenover et al.

    The stability of VNTRs is mainly associated with the length of repetitive unit, copy number of repetitive unit, and the purity Legendre et al. A program, serv , is now available for the selection and comparison of suitable VNTR for genotyping based on scoring the length of repetitive unit, copy number of repetitive unit, and the purity Legendre et al. Although MLVA is a rapid, easy to perform, inexpensive, and reproducible genotyping method with high resolution, VNTR loci may evolve too quickly to provide reliable phylogenetic relationships among closely related strains.

    Therefore, MLVA is unsuitable for long-term epidemiological surveillance, though it may be useful for tracking outbreaks of bacterial infections Lindstedt et al.

    The size difference in a VNTR locus may not always reflect the real number of tandem repeats because insertions or deletions in the amplified region can also give rise to the same size difference.

    Therefore, sequencing of the amplicons is necessary in this case. However, with the increase in the number of bacterial genome sequences available for a particular species, it may be possible to improve MLVA through the rational selection of suitable tandem repeat loci and primer design Johansson et al.

    In DGE, PCR amplicons with the same length are separated by electrophoresis in polyacrylamide gels in a sequence-dependent manner. The increasing gradient of denaturing components along the polyacrylamide gel opens double-stranded amplicons into single-stranded DNA through melting domains.

    The melting temperature of these domains is sequence-specific and decreases their mobility. Thus, different sequences will result in different origins of melting domains and consequently in different positions in the gel Muyzer et al.

    Such a gradient is obtained using either denaturing chemicals for denaturing gradient gel electrophoresis DGGE , or heat for temperature gradient gel electrophoresis TGGE and temporal temperature gradient electrophoresis TTGE Fromin et al.

    Applying statistical method makes DGE fingerprinting technique a promising tool Fromin et al. Numerous samples can be analyzed simultaneously, allowing the monitoring of microbial communities. This method consists of competitive quantitative PCR with group-specific primers targeting a variable region of 16S rRNA gene, followed by separation and quantification of the amplicons in the capillary containing high-molecular-weight linear polyacrylamide gel. CDCE can resolve sequences that differ by as little as a single base pair, and quantification of sequences is extremely sensitive due to the use of a laser-based detection system Lim et al.

    DGE has been widely used in exploring genetic diversity of bacteria since its first application in Muyzer et al. An advantage of DGGE is that selected bands can be sequenced. Thus, the presence of a particular phylotype can be monitored in the samples studied. However, DGGE requires a specific electrophoresis equipment and it is not possible to run several gels per run. The melting profile of a PCR product depending on its GC content, length, sequence, and heterozygosity, is conveniently monitored with saturating dyes that fluoresce in the presence of double-stranded DNA.

    HRM is not a banding pattern-based method sensu stricto but we classified it in this category of methods because it is based on PCR amplification and allows detection of sequence variants without sequencing or hybridization procedures Gundry et al.

    Using HRM, single nucleotide polymorphisms SNPs can be genotyped without probes and more complex regions can be typed with unlabeled hybridization probes. Using the clustered, regularly interspaced short palindromic repeats CRISPR locus of Campylobacter jejuni as target, HRM exhibited a discriminatory power comparable to that of the gold standard genotyping method, i.

    PFGE Price et al. The advantages of HRM are its homogeneity, rapidity 1—5 min , applicability to environmental samples, and the use of closed tubes. It has a similar or superior sensitivity and specificity to methods that require physical separation.

    In addition, comparability of results among laboratories may be limited. Because this method is a PCR-based genotyping method, it can be used with DNA taken directly from human specimens and environmental samples. In addition, the limited number of restriction fragments resulting from RE digestion of the PCR amplicons can be separated and visualized directly by gel electrophoresis without the need for probe hybridization.

    It has been used frequently for typing a variety of bacteria including Brucella species Al et al. Fragments are usually scored as either present or absent, and the presence or absence of bands in the second sample is referred to as polymorphism. Computer analysis of the number and size of fragments generated from each strain allows phylogenetic analysis Lindstedt et al. The discriminatory power of AFLP analysis is mainly determined by the combination of REs and the number of selective nucleotides in the primers.

    In silico analysis of bacterial genomic sequences can be used to select the most informative combinations of ERs and primer sequences Table 2 Keto-Timonen et al. Typically, 50— restriction fragments are coamplified in one fingerprint, depending on the complexity of the genome, the primers used, and the resolution of gel electrophoresis.

    Thus, relatively few primer pairs are used to visualize a large number of loci in AFLP analysis. It requires far less amount of DNA because multiple bands are derived from across the entire genome.

    The disadvantages of AFLP include the fact that automated analysis equipment may be required due to the many fragments involved and the huge quantity of information generated. DNA sequence, the nucleotide order in a genomic fragment, is the original genetic information of an organism and can be used directly for differentiation and phylogenetic analysis of bacterial strains.

    The most significant advantages of DNA sequencing-based genotyping over DNA banding pattern-based methods is its high reproducibility because it relies on unambiguous DNA sequences that can easily be stored in online databases and compared among laboratories.

    GenBank, the largest DNA sequence database, stores huge quantities of genomic sequences as well as locus-specific sequences from almost all known bacteria, and is the most frequently used sequence database by molecular microbiologists. DNA sequencing technology has profoundly influenced microbiology Salser, ; Hall, DNA sequencing-based genotyping of bacteria has significantly contributed to many aspects of genotyping by identifying SNPs, sequence deletions or insertions including sequence duplications, such as VNTRs , and genes under positive selection.

    Various types of differences among DNA sequences may be identified. This is the most common type of genetic variation Schork et al. In addition, deletion, insertion, and duplication events occur widely in bacterial genomes and are regarded as important evolutionary mechanisms. DNA sequencing is an efficient way to identify these genetic changes in order to differentiate and phylogenetically classify bacterial strains.

    In addition to detecting with a high sensitivity and specificity sequence differences, DNA sequencing may also allow the evaluation of the evolutionary forces that led to these differences. Relying on the neutral theory of molecular evolution, a high ratio of nonsynonymous over synonymous changes inferred from original DNA sequences can indicate positive selection, which confers a fitness benefit and will thus increase the frequency of that change Kimura, A well-known example of positive selection in action is the development of antibiotic resistance in bacteria.

    When exposed to antibiotics, most bacteria die quickly, but some may experience mutations that make them less susceptible to treatment. In addition, DNA sequencing-based identification of genes under positive selection is also an effective approach to identify virulence in bacteria Chen et al. Currently, there are two main strategies for DNA sequencing: The Sanger method is also referred to as dideoxy sequencing or chain termination DNA sequencing.

    In this method, the DNA sequence of a single-stranded template DNA is determined using DNA polymerase to synthesize a set of polynucleotide fragments of different lengths through the use of dideoxynucleotides that interrupt the elongation step of DNA amplification Sanger et al. Since its description in , the Sanger method has become the most widely used DNA sequencing method. However, recently, new DNA sequencing methods offering much higher throughput at lower cost have revolutionized DNA sequencing.

    Among these developments, pyrosequencing is the most remarkable. By comparison with pyrosequencing, Sanger sequencing is more expensive and requires more DNA quantity several micrograms and a cloning step for large sequencing projects such as genome sequencing.

    In contrast to the conventional Sanger method Sanger et al. The current state of pyrosequencing technology leads to an c. In , the Roche company first commercialized a high throughput sequencer using the pyrosequencing technology.

    Following shearing, genomic DNA fragments are hybridized to the surface of agarose beads, and then amplified by emulsion PCR and sequenced, without any cloning step Margulies et al. The current output of the Roche instrument is c. More recently, Illumina Solexa developed a pyrosequencer that uses glass-attached oligonucleotides that are complementary to specific adapters previously ligated onto DNA library fragments.

    Sequences are obtained by amplification using an isothermal polymerase, and Sanger-like sequencing. The Illumina method produces c. The latest pyrosequencing competitor is Applied Biosystems. Oligonucleotide adaptor-linked DNA fragments are coupled with magnetic beads that are covered with complementary oligonucleotides.

    To date, pyrosequencing has been used for several applications of genotyping. Sequencing the highly polymorphic regions in 16S rRNA gene by pyrosequencing demonstrated to be a rapid and inexpensive approach for identification and differentiation of bacteria Jonasson et al. In a recent article, Dethlefsen et al. Detection of antibiotic resistance has also benefited from pyrosequencing. Mutation detection in antibiotic resistance genes of M. Grouping, typing, and subtyping bacteria with specific virulence or of epidemiological interest may also be obtained.

    Pyrosequencing of genomic fragments has been used to genotype Neisseria gonorrhoeae and B. In comparison with Sanger sequencing, pyrosequencing, although being less expensive and having a higher output, is limited by short read lengths 25— bp , small numbers of samples that can be run simultaneously, and difficult sequence assembly. The ability to generate DNA sequences rapidly and the potential for scale-up make pyrosequencing a valuable option for SNP genotyping Clarke, At the strain level, however, 16S rRNA gene is too conserved to be useful, with the exception of few species such as N.

    In practice, 16S rRNA gene sequencing is mostly used for bacterial species identification. Some less-conserved genes, especially those under positive selection, have often been used for species identification and, in some rarer cases, for bacterial strain typing.

    Most of these genes encode bacterial surface proteins or virulence factors. The choice of appropriate genes may vary according to the species. DNA sequencing of the variable regions in the slpA gene, which encodes a surface layer protein of C. The set genes set 2, set 5, and set 7 , which encode S. Sequence analysis of the ompA gene, which encodes a major antigenic outer membrane protein of Rickettsia species, was demonstrated to be an important tool for identifying and subtyping these bacteria Fournier et al.

    Sequencing of the X region of the protein A gene spa is widely used for subtyping methicillin-resistant S. For more informative discrimination of MRSA strains, spa is strongly suggested to be combined with other gene loci or other genotyping methods Hallin et al.

    Although genes encoding surface proteins or virulence factors evolve very quickly, interpretation of the typing results may be misleading, especially in evaluating the bacterial population structure or in the case of a long-term epidemiological survey.

    Combination of genes encoding surface antigens or virulence factors with housekeeping genes may be a rational approach for bacterial strain typing, particularly for bacterial species with a high rate of genetic recombination Cai et al. MLST is a method using DNA sequencing to uncover allelic variants in several conserved genes usually seven genes , and is currently one of the most popular genotyping methods for characterizing bacterial strains Maiden et al. MLST examines multiple housekeeping genes whose sequences are constrained because of the essential function of the proteins they encode; the variation observed in these sequences is therefore neutral or nearly neutral.

    Typically, fragments of — bp of seven genes are sequenced and each different sequence for a given gene is attributed a number. Each strain is, therefore, assigned a seven-number allelic profile designated as sequence type ST Maiden et al. MLST is suitable for long-term investigation of bacterial population structures, particularly when subtyping bacterial species with a high rate of genetic recombination, such as N.

    DNA sequences are easily stored in online databases, which allow convenient exchange of strain typing data both within and between laboratories. So far, MLST has been applied to more than 23 bacterial species and is regarded as a reference genotyping method for many bacteria. Detailed information can be found on the MLST homepage, http: However, MLST also has drawbacks. First, alleles are assigned to a numbering system that is not representative of the actual gene sequence, which makes the phylogenetic analysis of tested strains poorly credible Clarke, Second, the use of highly conserved housekeeping genes in MLST often fails to detect the variability of closely related strains.

    Finally, sequencing of seven genes is costly and time consuming. ITS varies not only in sequence and length but also in the number of alleles and their positions on the chromosome Garcia-Martinez et al.

    While size variation of ITS resulting from either the amplification or digestion of amplicons by REs is useful for bacterial strain typing, sequencing ITS can provide a more comprehensive analysis of polymorphisms for strain typing. However, more than one copy of ITS is often found in the bacterial genome, and those ITS sequences within a given strain may differ from one another, making direct DNA sequencing of ITS in some bacterial species difficult.

    Cloning-based sequencing can resolve this problem but makes it a time-consuming method Garcia-Martinez et al. For the Bartonella genus, partial ITS amplification and sequencing has proven to be a sensitive tool for differentiating B. However, ITS is mainly used for species or subspecies identification, and less for strain typing. In addition, it is not suitable for bacteria from the order Rickettsiales because their 16s and 23S rRNA genes are not contiguous.

    MST, a novel genotyping system first applied to Y. MST assumes that noncoding DNA sequences, which are subject to less selection pressure than genes, vary more than genes and so are preferable for bacterial strain typing Drancourt et al.

    The intergenic spacers used as typing markers in MST are chosen to be those noncoding sequences varying the most between aligned genomes of bacterial strains within one species, or between closely related species if only one genome sequence is available for a given species Drancourt et al. For example, the genome sequences of the closely related B. This work demonstrated the great potential of MST for bacterial strain typing. With rapid accumulation of bacterial genome sequences and the increasing number of species with more than two genome sequences available, the highly variable intergenic spacers selected by genome comparison are becoming more representative and powerful for bacterial strain typing.

    The phylogenetic organization of studied strains, however, was inferred from concatenation of all selected intergenic spacers Fig. MST has been applied successfully to several human pathogens, including Y. Indeed, MST has been successful in subtyping Y. Recently, MST was also used to evaluate the genetic diversity of B.

    Another advantage of MST is that primers can be chosen in conserved regions of the flanking genes, making amplification of highly variable spacers easy. While intergenic spacers are considered as rapidly evolving markers, MST has succeeded in establishing reliable correlations of MST genotypes with geographic distribution, clinical manifestations, and epidemiology of strains Drancourt et al.

    In addition, MST enabled retrospective analysis of Y. A recent study applying MST to B. An online database, MST-Rick, which provides a local blast program, enables anyone to compare his own spacer sequences and determine MST genotypes Table 2.

    By giving access to the complete genetic content of bacterial strains, genome sequencing is the ultimate genotyping method. Both the Sanger and pyrosequencing methods are currently used for genome sequencing, either separately or combined. The former technology has been the mostly used worldwide since the genome of Haemophilus influenzae was sequenced in Fleischmann et al. Genome sequencing using the Sanger method requires the construction of a cDNA library and produces —bp reads, with a maximum output of 0.

    In contrast, pyrosequencing produces short reads 25— bp but with an output of up to 3—4 Gb per run. Because of the short read length, making sequence assembly difficult, pyrosequencing may currently be preferred for genome resequencing than for de novo assembly, although a combination of Sanger sequencing and next generation sequencing has been proposed as a valuable alternative for this latter purpose.

    Recently, pyrosequencing was used for rapid genome sequencing of F. Pyrosequencing also proved to be useful for metagenomic studies Eckburg et al. However, although sequencing techniques have been greatly improved since the first bacterial genome was sequenced in Fleischmann et al. DNA hybridization is widely used for detecting DNA mutations because it requires sequence complementarity to occur. Two main elements, probes and targets, are involved in DNA hybridization. Probes are DNA fragments of known sequences, whereas targets are the free nucleic acids whose identity and abundance are detected by hybridization with fluorescently labeled probes based on their complementarity to the probes.

    A DNA array enables genomic DNA to be tested for its ability to hybridize to hundreds or tens of thousands of DNA fragments or oligonucleotides spots arrayed on a substrate. Substrates can be membranes, glass, plastic, silicon wafers, or metal alloys. An array is a very powerful tool for studying the transcriptosome as well as the genetic diversity of bacteria. Two classes of DNA arrays, macroarrays and microarrays, that differ in the size and number of spots on the supports are used.

    Macroarrays are usually printed on nylon membranes c. A microarray, in contrast, is much denser than a macroarray, with up to 1 spots per array. Macroarrays offer a rapid, specific, and cost-efficient genotyping method without the need to purchase expensive equipment, and has proven particularly effective for detecting genes involved in antibiotic resistance Johansen et al.

    In a recent study, one group rapidly detected mutations in rpoB, katG, inhA , and ahpC , which are associated with rifampin and isoniazid resistance in M. The macroarray approach in this study showed high sensitivity and specificity and could even be directly applied to DNA extracted from clinical specimens Zhang et al.

    Spoligotyping, detection of the polymorphism in the direct variant repeats DVRs loci of the mycobacterial chromosome, is a PCR-based macroarray technique for simultaneous detection and differentiation of MTC bacteria Groenen et al.

    Amplification of the DVRs using primers complementary to the DRs, followed by the hybridization of amplicons to the oligonucleotides derived from the spacer sequences probes , generates distinct spoligotyping patterns. Spoligotyping has been proven to be a rapid, reproducible, informative, and effective technique for use on DNA taken directly from human specimens or environmental samples without the need for prior culture. It has been widely used in epidemiological studies of M.

    An international spoligotyping database, SpolDB4, suggested the existence of geographical genetic clones within MTC populations and provided a large-scale conceptual framework for the global TB Epidemiologic Network Brudey et al.

    For Corynebacterium diphtheriae , a macroarray-based spoligotyping method based on hybridization analysis of two CRISPR loci differentiated 20 strains into three spoligotypes Mokrousov et al. By comparison with microarray, DNA macroarray is less expensive but suffers from a lower discriminatory power due to the small number of characters studied.

    DNA microarrays are specially treated microscope slides chips that carry an ordered mosaic of sequences representing most or all of the genes of an organism. Since its development in Schena et al.

    In contrast to Southern blotting and macroarray techniques, where a limited number of probes are transferred onto nitrocellulose or nylon membranes, a DNA microarray includes tens of thousands of probes arrayed directly on the support and it therefore constitutes a high-throughput genotyping method Garaizar et al.

    However, compared with other locus-limited genotyping methods, the DNA microarray remains more expensive. In addition, satisfactory quality controls are difficult to achieve in DNA microarray analysis because many factors affect nucleic acid hybridization reactions.

    The probe design in a DNA microarray is mainly based on reference DNA sequences, including prior knowledge of the polymorphisms derived from other genotyping studies.

    As a result, DNA microarray analysis may underestimate the genetic diversity among the bacterial population. There are two kinds of DNA microarrays: A cDNA microarray uses cDNA as probes and is often used to identify the presence or absence of genes in a technique called comparative genomic hybridization.

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    This app is only available on the App Store for iOS devices. Tweaked small aspects of the pickup and menu user experience. Home screen updates and fixes with improved location handling. Design and format continuity between images displayed within the app. Improvements to the map for a smoother experience. Other minor bug fixes and improvements. Fixed an issue that was rejecting logins and signups.

    Fixed a bug where dispensaries could be displayed as closed during open times. Deals now featured on the home screen. Minor fixes and performance updates. Fixes a crash for some users while browsing the home screen. We've made some behind the scenes changes to support our neighbors to the North. And for everyone's enjoyment, we have made performance improvements to menus along with other minor fixes and updates.

    Enjoy our brand new home screen experience! Bug Fixes When leaving a review, a sign-in request will now consistently appear to ensure that your words of wisdom can be shared with others. Also, if you couldn't even get a review to submit, we've got a fix for you as well! Tapping thumbs down on someone's review will now not only let the world know you disapprove, but also show a number other than negative one. Checking out deals will now give you a one time we promise we won't ask you again option to turn on notifications to be alerted to new promotions.

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    Jack Herer Strain (2019 Review)

    Mar 2, User-generated content (UGC) allows brands to interact through personalized content — real-time chat, online Go to the profile of Kelly Strain. Sep 17, They also differ vastly in their reported effects on users (more on that below). strain of cannabis plant produces a flower with a unique cannabinoid profile. Hybrid strains are cannabis strains created by crossbreeding. Jan 9, Check out our complete, in-depth Jack Herer marijuana strain review sativa hybrid including info on flavor profile, growing, and medicinal Said to be a well- generated blend between its parent strains Shiva .. Term of Use.

    Amnesia Haze Strain Review: Aroma, Flavor, and Appearance



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    Spartak7777

    Mar 2, User-generated content (UGC) allows brands to interact through personalized content — real-time chat, online Go to the profile of Kelly Strain.

    Rinaldinio

    Sep 17, They also differ vastly in their reported effects on users (more on that below). strain of cannabis plant produces a flower with a unique cannabinoid profile. Hybrid strains are cannabis strains created by crossbreeding.

    fors

    Jan 9, Check out our complete, in-depth Jack Herer marijuana strain review sativa hybrid including info on flavor profile, growing, and medicinal Said to be a well- generated blend between its parent strains Shiva .. Term of Use.

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