Molecular Tools For Detection Research Articles

Field deployment of loop-mediated isothermal amplification for centralized mass-screening of asymptomatic malaria in Zanzibar: a pre-elimination setting.

BackgroundMolecular tools for detection of low-density asymptomatic Plasmodium infections are needed in malaria elimination efforts. This study reports results from the hitherto largest implementation of loop-mediated isothermal amplification (LAMP) for centralized mass screening of asymptomatic malaria in Zanzibar.MethodsHealthy individuals present and willing to participate in randomly selected households in 60 villages throughout Zanzibar were screened for malaria by rapid diagnostic tests (RDT). In 50 % of the study households, participants were asked to provide 60 μL of finger-prick blood for additional LAMP screening. LAMP was conducted in two centralized laboratories in Zanzibar, by trained technicians with limited or no previous experience of molecular methods. The LAMP assay was performed with LoopampTM MALARIA Pan/Pf Detection Kit (Eiken Chemical Company, Japan). Samples positive for Plasmodium genus (Pan)-LAMP were re-tested using Plasmodium falciparum-specific LAMP kits.ResultsPaired RDT and LAMP samples were available from 3983 individuals. The prevalence of asymptomatic malaria was 0.5 % (CI 95 % 0.1-0.8) and 1.6 % (CI 95 % 1.1-2.2) by RDT and Pan-LAMP, respectively. LAMP detected 3.4 (CI 95 % 2.2-5.2) times more Plasmodium positive samples than RDT. DNA contamination was experienced, but solved by repetitive decontamination of all equipment and reagents.ConclusionsLAMP is a simple and sensitive molecular tool, and has potential in active surveillance and mass-screening programmes for detection of low-density asymptomatic malaria in pre-elimination settings. However, in order to deploy LAMP more effectively in field settings, protocols may need to be adapted for processing larger numbers of samples. A higher throughput, affordable closed system would be ideal to avoid contamination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0731-2) contains supplementary material, which is available to authorized users.

Application of real-time PCR in pathogenic detection of necrotizing herpes stromal keratitis

Background Herpes stromal keratitis (HSK) is a common infectious ocular surface disease, with a higher recurrent rate, especially in necrotizing HSK.The diagnosis of HSK primarily depends on signs and symptoms, and specific laboratory diagnostic is lack. Objective This study was to clarify the expression of herpes simplex virus (HSV) in the corneal epithelium scrapings and tears of necrotizing HSK patients. Methods Thirty eyes of 30 patients with necrotizing HSK were enrolled in Nanjing Hospital of Nanjing Medical Hospital from September 2012 to September 2013 under the patient's informed consent.The eyes were examined by slit lamp microscope and scored.HSK patients received local and systemic therapy for 8 weeks and then an oral maintenance dose for 6 months.Corneal epithelial scrapings and tears samples were collected for HSV DNA detection by real-time PCR before and the 1st, 2nd, 4th, 6th and 8th week after therapy respectively.The difference of HSV positive rate was compared between corneal epithelium scrapings and tears samples using Chi-square test.Multilevel mixed effective model was employed to evaluate HSV concentration change in the samples at various time points in the HSV-positive patients of initial visit.The correlation between HSV concentration and clinical score was analyzed by Spearman rank correlation. Results HSV-positive rate was 46.4% (13/30) in the corneal epithelial scrapings and 13.3% (4/30) in the tear samples, showing a significant difference between them (P=0.006). HSV-positive rate was significantly lower in the corneal epithelial scrapings 1 week, 2 weeks and 4 weeks after treatment than before (P=0.001, 0.003, 0.004), and no HSV was detected 6 weeks and 8 weeks after treatment.No significant change in HSV-positive rate in tear samples in 1 week and 2 weeks after treatment in comparison with before treatment (P=1.000, 0.583), and no HSV was detected after 4 weeks following treatment.The HSV concentration was 2 460(2 165-636 500)/ml in initial 13 HSV-positive eyes of corneal epithelial scrapings and 0(0-1 150)/ml in initial 13 HSV-positive eyes of tear samples.Multilevel mixed effective model determined that HSV concentration was significantly lower in corneal epithelial scraping than that in tear (P=0.005), and HSV concentration was reduced with the lapse of time (P=0.001), with the faster rate of decline in the corneal epithelial scrapings (P=0.049). A positive correlation was found between initial HSV concentration and clinical scores (rs=0.844, P=0.000). Conclusions Real-time PCR appears to be a powerful molecular tool for the detection of HSV in the HSK, especially in corneal epithelial scrapings of lesion.The initial positive outcome of viral DNA in corneal epithelial scrapings predicts a severe clinical procedure. Key words: Herpes simplex/diagnosis; Corneal stroma; Keratitis; Necrosis; DNA, viral/analysis; Epithelium/virology; Tears/virology; Real-time PCR

Molecular diagnosis of Fragile X syndrome in subjects with intellectual disability of unknown origin: implications of its prevalence in regional Pakistan.

Fragile-X syndrome (FXS) is the most common form of inherited intellectual disability (ID) and affects 0.7–3.0% of intellectually compromised population of unknown etiology worldwide. It is mostly caused by repeat expansion mutations in the FMR1 at chromosome Xq27.3. The present study aimed to develop molecular diagnostic tools for a better detection of FXS, to assess implementation of diagnostic protocols in a developing country and to estimate the prevalence of FXS in a cohort of intellectually disabled subjects from Pakistan. From a large pool of individuals with below normal IQ range, 395 subjects with intellectual disability of unknown etiology belonging to different regions of the country were recruited. Conventional-PCR, modified-PCR and Southern blot analysis methods were employed for the detection of CGG repeat polymorphisms in the FMR1 gene. Initial screening with conventional-PCR identified 13 suspected patients. Subsequent investigations through modified PCR and Southern blot analyses confirmed the presence of the FMR1 mutation, suggesting a prevalence of 3.5% and 2.8% (mean 3.3%) among the male and female ID patients, respectively. These diagnostic methods were further customized with the in-house conditions to offer robust screening of referral patients/families for diagnostics and genetic counseling. Prescreening and early diagnosis are crucial for designing a prudent strategy for the management of subjects with ID. Outcome of the study recommends health practitioners for implementation of molecular based FXS diagnosis in routine clinical practice to give a better care for patients similar to the ones included in the study.

The draft genome sequence of Xanthomonas species strain Nyagatare, isolated from diseased bean in Rwanda.

We announce the genome sequence for Xanthomonas species strain Nyagatare, isolated from beans showing unusual disease symptoms in Rwanda. This strain represents the first sequenced genome belonging to an as-yet undescribed Xanthomonas species known as species-level clade 1. It has at least 100 kb of genomic sequence that shows little or no sequence similarity to other xanthomonads, including a unique lipopolysaccharide synthesis gene cluster. At least one genomic region appears to have been acquired from relatives of Agrobacterium or Rhizobium species. The genome encodes homologues of only three known type-three secretion system effectors: AvrBs2, XopF1 and AvrXv4. Availability of the genome sequence will facilitate development of molecular tools for detection and diagnostics for this newly discovered pathogen of beans and facilitate epidemiological investigations of a potential causal link between this pathogen and the disease outbreak.

Functional Siglec lectin domains from soluble expression in the cytoplasm of Escherichia coli

Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of mammalian cell-surface receptors that are involved in cell–cell interactions and signaling functions, primarily expressed on cells of the immune system. Key to their function is their specific binding of distinct sialylated glycan ligands mediated via an N-terminal carbohydrate recognition (lectin) domain. Studies concerning the molecular basis of their individual carbohydrate specificities are rare due to the absence of suitable recombinant expression methods for producing these disulfide-containing proteins in sufficient quantities required for their in-depth in vitro characterization. We established an efficient E. coli-based expression and purification method for Siglec lectin domains, utilizing the trxB gor suppressor strain Rosetta-gami B (DE3) in which proper folding with intact disulfide bonds was achieved in the cytoplasm. The approach is demonstrated for human Siglec-7, -8 and -9 lectin domains and works equally well for expression in nutrient-rich (LB) or minimal growth medium, allowing stable-isotope labeling for NMR studies. The recombinant proteins were properly folded as proven by 2D 1H–15N HSQC NMR spectroscopy and by thermal unfolding followed by CD spectroscopy, and functionally active as confirmed by monitoring ligand binding using NMR titration experiments. Our method enables efficient production of homogeneous and active protein samples in milligram quantities. Its implementation will significantly enhance future structure–function studies of this important class of immune-modulating receptors and will support a variety of applications including screening for natural and synthetic ligands or the development of fluorescently-labeled molecular tools for glycan ligand detection or flow-cytometric cell sorting.

Partial characterization and development of sensitive and reliablediagnostic for the detection of cucumber mosaic virus

A survey on Cucumber mosaic virus (CMV) was conducted on 21 banana orchards in three bananas producing states: Karnataka, Maharashtra, and Uttar Pradesh. Sampling was done during winter and autumn and all samples were initially examined for the presence of CMV using DAS-ELISA. Out of 300 tested samples, the presence of CMV was observed in 13. The lowest rate of infection was determined in Uttar Pradesh followed by Maharashtra, while high infections were observed in samples from Karnataka. Three CMV infected samples represented in each state were amplified through RT-PCR using the movement protein (MP) gene specific primers and cloned in the pGEMT essay vector. The RT-PCR products of amplified samples were sequenced. Gene sequences of CMV-KAR, CMV-MR, and CMV-UP isolates under study coding for the movement protein revealed 99.76%-99.88% and 99.28%-99.64% sequence identity at the nucleotide and amino-acid level. These isolates were compared with 29 CMV isolates reported from various plants around the world, which formed three distinct subclades: -IA, -IB, and -II. These isolates were most closely related to subgroup-I isolates, sharing up to 95.81% and 96.84% sequence identity at nucleotide and amino acid, respectively. However, these isolates shared 81.79%-81.90% and 82.80%-83.15% nucleotide and amino acid identity with subgroup II isolates. The isolates under study clustered with the CMV subgroup-IB as confirmed by phylogenetic analysis. Further, we standardized the minimum limit of RNA transcript for the sensitivity of RT-PCR with respect to the coat protein (CP) gene and movement protein (MP) gene. The results of RT-PCR showed that the CP gene was more sensitive than the MP gene for the detection of CMV as it amplified the gene product up to 0.02 ng RNA. The CP gene based RT-PCR assay may be a more sensitive, reliable, and convenient molecular tool for detection of the CMV pathogen, and can be used in quarantine, eradication, and tissue culture certification programs.

Past and Present Therapeutics for Alzheimer’s Disease

Alzheimer’s disease is a progressive, degenerative disorder that attacks the brain’s nerve cells, or neurons, resulting in loss of memory, thinking and language skills, and behavioural changes. Alzheimer’s disease is the most common cause of dementia, or loss of intellectual function, among people aged 65 and older. Two types of abnormal lesions clog the brains of individuals with Alzheimer’s disease: Beta-amyloid plaques-sticky clumps of protein fragments and cellular material that form outside and around neurons; and neurofibrillary tangles-insoluble twisted fibers composed largely of the protein tau that build up inside nerve cells. Several molecular biomarkers are recognised to be associated with the pathology of AD. However, the currently available laboratory methods are not sensitive enough for such an early diagnosis. Nanotechnology offers a number of highly sensitive molecular detection tools that may make this goal achievable. These nanodiagnostic tools utilise different nanoparticles/nanostructures, statins and are based on different physicochemical interactions that may be utilised either in vitro or in vivo.

Molecular diagnostic tools for detection and differentiation of phytoplasmas based on chaperonin-60 reveal differences in host plant infection patterns.

Phytoplasmas (‘Candidatus Phytoplasma’ spp.) are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S–23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (cpn60) gene sequence from a diverse array of ‘Ca.Phytoplasma’ spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from ‘Ca.Phytoplasma’ spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns.

Newly identified host range of European mountain ash ringspot‐associated virus (EMARaV) and its distribution in the Czech Republic

SummaryIt has been previously shown that European mountain ash ringspot‐associated virus (EMARaV), a multipartite, negative‐strand RNA plant virus, is pathogenic for European mountain ash (Sorbus aucuparia L.). However, its transmissibility to and replication in other host species remains poorly understood. In this work, we performed experimental inoculations of seventeen virus‐tested species and interspecies hybrids of subfamily Maloideae (family: Rosaceae) with buds from EMARaV‐infected S. aucuparia trees. Surprisingly, a majority of the graft‐inoculated tree species, for instance Sorbaronia, Aronia and Amelanchier, developed symptoms typical for EMARaV infection. These symptoms included chlorotic spots of various size and shape and mottling on leaves but not on flowers and fruits. The propagation of EMARaV in graft‐inoculated tree species was subsequently confirmed by molecular detection tools. Based on these results, we carried out a systematic survey throughout the Czech Republic and identified the presence of EMARaV in wild growing trees in all explored regions, with massive disease infestation in several locations. More importantly, EMARaV infection was newly detected in S. aucuparia subsp. moravica, S. aucuparia × Crataegus sanguinea cv. Granatnaja, (Sorbus aria × Aronia arbutifolia) × S. aucuparia cv. Burka, cv. Finskaja (a Sorbus hybrid of unknown origin) and Aronia melanocarpa; all these EMARaV‐infested trees were grown in production orchards. The sequencing of the nucleocapsid (NC)‐coding gene from several of these EMARaV isolates provided insight into the population structure and phylogenetic relationships of the EMARaV variants occurring in the Czech Republic. Together, our biological and molecular findings extend our knowledge on the host range of EMARaV and provide the first evidence of its natural occurrence in hosts dissimilar to S. aucuparia.

Clinical and molecular investigation of johne’s disease among small ruminants in Makkah, Saudi Arabia

Paratuberculosis (PTB) or Johne’s disease (JD) is a chronic debilitating disease caused by the Mycobacterium avium subsp . paratuberculosis (MAP). JD affects a wide range of animals including ruminants and characterized by enteritis and progressive diarrhea. The current study aimed to implement molecular tools for detection and identification of MAP among clinically suspected small ruminants in Makkah region, Kingdom of Saudi Arabia (KSA). A total of 2660 small ruminants from five different farms around Makkah were clinically investigated for characteristic signs of JD during the period of November 2013 to February of 2014. Out of investigated animals, 16 cases were selected as being suspected of JD infection based on the associated clinical symptoms (emaciation and unthriftiness with or without persistent diarrhea). Rectal scrapings were collected from all suspected animals and were subjected for molecular examination. Three different genetic targets were evaluated including 16S rDNA, insertion sequence 900 (IS900) and intergenic spacer (IGS). Initial examination of suspected specimens by amplification of universal bacterial primers for 16S rDNA revealed positive results in 6 out of 16 cases (37.5%). Further amplification of the Mycobacterium -specific IGS target from 16s rDNA-positive samples revealed the detection of 4 Mycobacterium species. Sequence analysis of the IGS sequence of these 4 Mycobacterium species revealed 2 cases only as Mycobacterium avium paratuberculosis . This finding was confirmed by the detection of MAP-specific IS900 target only from the same 2 cases. In conclusion, the current study genetically documented the first report of MAP (the causative agent of JD) among small ruminants of Makkah region.

Real-time PCR detection and discrimination of the Ceratocystis coerulescens complex and of the fungal species from the Ceratocystis polonica complex validated on pure cultures and bark beetle vectors

Eight Ceratocystis Ellis & Halst. species belonging to the Ceratocystis coerulescens complex are pathogens causing blue-stain on Pinaceae. Three of these species, namely C. polonica, C. laricicola, and C. fujiensis, are particularly aggressive and can cause tree mortality. Although currently absent from the North American landscape, they are considered a significant potential threat to the Canadian boreal forest. As they are difficult to distinguish from native North American species belonging to the C. coerulescens complex, we developed a real-time PCR detection test for each of the three species to detect the equivalent of one fungal spore directly from insect vectors. DNA from at least one species of the C. coerulescens complex was detected on 86% of the beetles (Ips typographus (Linnaeus, 1758) and Ips cembrae (Heer, 1836)), whereas C. polonica DNA was detected on 60% of the I. typographus and C. laricicola DNA was detected on 84% of the I. cembrae. Between 20 and 344 225 spore equivalents were detected on the beetle specimens, and no inhibition effect of DNA extract from environmental samples was observed. These molecular detection tools will allow for rapid and reliable detection of C. polonica, C. laricicola, or C. fujiensis, allowing for a rapid implementation of eradication measures in case of introduction into Canada.

Synthesis and characterization of a glycine-modified heptamethine indocyanine dye for in vivo cancer-targeted near-infrared imaging.

Near-infrared (NIR) fluorescent sensors have emerged as promising molecular tools for cancer imaging and detection in living systems. However, cancer NIR fluorescent sensors are very challenging to develop because they are required to exhibit good specificity and low toxicity as an eligible contrast agent. Here, we describe the synthesis of a new heptamethine indocyanine dye (NIR-27) modified with a glycine at the end of each N-alkyl side chain, and its biological characterization for in vivo cancer-targeted NIR imaging. In addition to its high specificity, NIR-27 also shows lower cytotoxicity than indocyanine green, a nonspecific NIR probe widely used in clinic. These characteristics suggest that NIR-27 is a promising prospect as a new NIR fluorescent sensor for sensitive cancer detection.

Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay

Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV.

A molecular tool for detection and tracking of a potential indigenous Beauveria bassiana strain for managing emerald ash borer populations in Canada

Emerald ash borer is an invasive species from Asia. Beauveria bassiana strain L49–1AA is being tested for the control of emerald ash borer in Canada, using an autocontamination trapping system. We have developed a simplified allele discrimination polymerase chain reaction (PCR) assay to screen B. bassiana strain, L49–1AA from other Beauveria species by targeting the inter-strain genetic differences in 5′ end of EF1-α gene of the genus Beauveria. A single nucleotide polymorphism (SNP) site, T→C was identified only in L49–1AA and was used to develop a simplified allele discrimination polymerase chain reaction (PCR) assay based on a modified allelic inhibition of displacement activity (AIDA) approach for distinguishing B. bassiana L49–1AA from all background Beauveria isolates. The SNP site was employed to design inner primers but with a deliberate mismatch introduced at the 3′ antepenultimate from the mutation site in order to maximize specificity and detection efficiency. Amplification was specific to L49–1AA without cross-reaction with DNA from other Beauveria strains. In addition, the designed primers were also tested against environmental samples in L49–1AA released plots and observed to be highly efficient in detecting and discriminating the target strain, L49–1AA from both pure and crude DNA samples. This new method can potentially allow for more discriminatory tracking and monitoring of released L49–1AA in our autocontamination and dissemination projects for managing EAB populations. Additionally, the modified-AIDA format has potential as a tool for simultaneously identifying and differentiating closely related Beauveria species, strains/isolates as well as general classification of other pathogens or organisms.

A sensitive and specific molecular tool for detection of both typical and atypical enteroaggregative Escherichia coli

A multiplex PCR has been standardized and evaluated for the identification of both typical and atypical enteroaggregative Escherichia coli (EAEC). The assay detecting aaiA, aaiG, aggR and aatA presented 94.8% of sensitivity, 94.3% of specificity and was able to efficiently detect both groups of EAEC among E. coli isolated from stool cultures.

Comparison of peptide nucleic acid fluorescence in situ hybridization assays with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry for the identification of bacteria and yeasts from blood cultures and cerebrospinal fluid cultures

Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a molecular diagnostic tool for the rapid detection of pathogens directly from liquid media. The aim of this study was to prospectively evaluate PNA FISH assays in comparison with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, as a reference method, for both blood and cerebrospinal fluid (CSF) cultures, during a 1-year investigation. On the basis of the Gram stain microscopy results, four different PNA FISH commercially available assays were used (‘Staphylococcus aureus/CNS’, ‘Enterococcus faecalis/OE’, ‘GNR Traffic Light’ and ‘Yeasts Traffic Light’ PNA FISH assays, AdvanDx). The four PNA FISH assays were applied to 956 positive blood cultures (921 for bacteria and 35 for yeasts) and 11 CSF cultures. Among the 921 blood samples positive for bacteria, PNA FISH gave concordant results with MALDI-TOF MS in 908/921 (98.64%) samples, showing an agreement of 99.4% in the case of monomicrobial infections. As regards yeasts, the PNA FISH assay showed a 100% agreement with the result obtained by MALDI-TOF MS. When PNA FISH assays were tested on the 11 CSF cultures, the results agreed with the reference method in all cases (100%). PNA FISH assays provided species identification at least one work-day before the MALDI-TOF MS culture-based identification. PNA FISH assays showed an excellent efficacy in the prompt identification of main pathogens, yielding a significant reduction in reporting time and leading to more appropriate patient management and therapy in cases of sepsis and severe infections.

Ultrasensitive detection and identification of BRAF V600 mutations in fresh frozen, FFPE, and plasma samples of melanoma patients by E-ice-COLD-PCR.

A number of molecular diagnostic methods have been developed for the detection and identification of mutations in tumor samples, which are important for the choice of treatment in the context of personalized medicine. For the treatment of metastatic melanoma, Vemurafenib is recommended for patients with BRAF V600 activating mutations. However, the different assays developed to date for the detection of these mutations lack sensitivity or specificity or do not allow a sequencing-based identification or validation of the mutation. Recently, enhanced improved and complete enrichment co-amplification at lower denaturation temperature-polymerase chain reaction (E-ice-COLD-PCR) has been developed as a sensitive method for the detection and identification of mutations in KRAS codons 12/13. Here, we present the first E-ice-COLD-PCR assay for the detection and identification of BRAF codon 600 mutations, which has a large dynamic range, as 25pg to 25ng can be used as DNA input without any reduction in mutation enrichment efficiency, and which can detect down to 0.01% of mutated alleles in a wild-type background. The assay has been validated on fresh frozen, formalin-fixed paraffin-embedded (FFPE), and plasma samples of melanoma patients and has allowed the detection and identification of BRAF mutations present in samples appearing as wild type using standard pyrosequencing, endpoint genotyping, or Sanger sequencing. Thus, the BRAF V600 E-ice-COLD-PCR assay is currently one of the most powerful molecular diagnostic tools for the ultrasensitive detection and identification of BRAF codon 600 mutations.

Bacteriophages of leuconostoc, oenococcus, and weissella.

Leuconostoc (Ln.), Weissella, and Oenococcus form a group of related genera of lactic acid bacteria, which once all shared the name Leuconostoc. They are associated with plants, fermented vegetable products, raw milk, dairy products, meat, and fish. Most of industrially relevant Leuconostoc strains can be classified as either Ln. mesenteroides or Ln. pseudomesenteroides. They are important flavor producers in dairy fermentations and they initiate nearly all vegetable fermentations. Therefore, bacteriophages attacking Leuconostoc strains may negatively influence the production process. Bacteriophages attacking Leuconostoc strains were first reported in 1946. Since then, the majority of described Leuconostoc phages was isolated from either dairy products or fermented vegetable products. Both lytic and temperate phages of Leuconostoc were reported. Most of Leuconostoc phages examined using electron microscopy belong to the Siphoviridae family and differ in morphological details. Hybridization and comparative genomic studies of Leuconostoc phages suggest that they can be divided into several groups, however overall diversity of Leuconostoc phages is much lower as compared to, e.g., lactococcal phages. Several fully sequenced genomes of Leuconostoc phages have been deposited in public databases. Lytic phages of Leuconostoc can be divided into two host species-specific groups with similarly organized genomes that shared very low nucleotide similarity. Phages of dairy Leuconostoc have rather limited host-ranges. The receptor binding proteins of two lytic Ln. pseudomesenteroides phages have been identified. Molecular tools for detection of dairy Leuconostoc phages have been developed. The rather limited data on phages of Oenococcus and Weissella show that (i) lysogeny seems to be abundant in Oenococcus strains, and (ii) several phages infecting Weissella cibaria are also able to productively infect strains of other Weissella species and even strains of the genus Lactobacillus.

High‐resolution melting analysis for rapid detection and characterization of Botrytis cinerea phenotypes resistant to fenhexamid and boscalid

A novel, high‐resolution melting (HRM) analysis was developed to detect single nucleotide polymorphisms (SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty‐six single‐spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine (F) with serine (S) or valine (V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdhB gene resulted in the replacement of histidine (H) with arginine (R) or tyrosine (Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high‐resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B. cinerea populations.

Transcriptomic Architecture of the Adjacent Airway Field Cancerization in Non–Small Cell Lung Cancer

Earlier work identified specific tumor-promoting abnormalities that are shared between lung cancers and adjacent normal bronchial epithelia. We sought to characterize the yet unknown global molecular and adjacent airway field cancerization (FC) in early-stage non-small cell lung cancer (NSCLC). Whole-transcriptome expression profiling of resected early-stage (I-IIIA) NSCLC specimens (n = 20) with matched tumors, multiple cytologically controlled normal airways with varying distances from tumors, and uninvolved normal lung tissues (n = 194 samples) was performed using the Affymetrix Human Gene 1.0 ST platform. Mixed-effects models were used to identify differentially expressed genes among groups. Ordinal regression analysis was performed to characterize site-dependent airway expression profiles. All statistical tests were two-sided, except where noted. We identified differentially expressed gene features (n = 1661) between NSCLCs and airways compared with normal lung tissues, a subset of which (n = 299), after gene set enrichment analysis, statistically significantly (P < .001) distinguished large airways in lung cancer patients from airways in cancer-free smokers. In addition, we identified genes (n = 422) statistically significantly and progressively differentially expressed in airways by distance from tumors that were found to be congruently modulated between NSCLCs and normal lung tissues. Furthermore, LAPTM4B, with statistically significantly increased expression (P < .05) in airways with shorter distance from tumors, was upregulated in human immortalized cells compared with normal bronchial epithelial cells (P < .001) and promoted anchorage-dependent and -independent lung cancer cell growth. The adjacent airway FC comprises both site-independent profiles as well as gradient and localized airway expression patterns. Profiling of the airway FC may provide new insights into NSCLC oncogenesis and molecular tools for detection of the disease.