Amplicon Size Research Articles

Molecular Identification of Rice Brown Leaf Spot Fungus Using Conserved Region of Glycoside hydrolase Gene

Rice brown leaf spot disease is one of the most devastating fungal diseases that affect rice plants and reduces the yield across the globe. In the present study, twenty isolates from different rice-growing districts in Tamil Nadu by tissue segment approach was isolated from disease-infected leaves and a purified. The isolates were identified at the genus level using ITS primers, which resulted in an amplicon size of a 550 bp. With the use of species-specific primers of BF and BR the isolate was confirmed as B. oryzae. Gene-specific primers were designed using Primer 3 plus software for the Glycoside hydrolase family 13 protein, which is a highly conserved gene in B. oryzae. All isolates were sequenced using gene-specific primers, and phylogenetic tree analysis was used to investigate the isolate's diversity. The results revealed considerable variance among the B. oryzae collected from different locations of Tamil Nadu with other B .oryzae isolates retrieved from the NCBI database. The isolates were phenotypically validated using morphological and cultural features

ALLELIC VARIATIONS OF SOS, HVNHX AND SDO GENES IN SOME BARLEY (Hordeum Vulgare L.) GENOTYPES

This study has been carried out at the Biotechnology Lab., Department of Field Crops, Faculty of Agriculture, Damascus University during the growing season 2019 - 2020, in order to detect the variations of SOS, HVNHX and SDO genes in different barley genotypes. Clear variations in the SOS, HVNHX and SDO genes, which are responsible for salinity tolerance were found among the investigated genotypes. It has been found that the variation in the amplicon size between loci per gene was very high in some cases, while there was a high degree of symmetry in other cases, and could be easily distinguished on 2% Agarose gel. The PCR results for the SOS genes (SOS1, SOS2, SOS3), HVNHX genes (HVNHX1, HVNHX2, HVNHX3) and SDO genes (Cu/Zn SODII, Cu/Zn SODI, CAT, GRI , APXIII) have shown only one morphological pattern in most of the studied genotypes, while revealed two patterns for the SOS3 gene, but the rest of genes (HVNHX1, HVNHX2, HVNHX3 Cu/Zn SODI, CAT) exhibited only one morphological pattern. The SOS3 was superior in the number of polymorphic patterns, as the number of total patterns was 14 in all the studied genotypes, but the Cu/Zn SODI showed the least number of polymorphic patterns with only 1 pattern, while the largest number (7 patterns) was detected in the genotype (H9), but the two genotypes Fourat9 and Fourat7 showed only one polymorphic pattern.

ACC deaminase producing plant growth promoting rhizobacteria enhance salinity stress tolerance in Pisum sativum.

The online version contains supplementary material available at 10.1007/s13205-021-03047-5.

Rapid PCR-based method for herbivore dietary evaluation using plant-specific primers.

Polyphagous pests cause significant economic loss worldwide through feeding damage on various cash crops. However, their diets in agricultural landscapes remain largely unexplored. Pest dietary evaluation in agricultural fields is a challenging task currently approached through visual observation of plant feeding and microscopic identification of semi-digested plant material in pest’s guts. While molecular gut content analysis using metabarcoding approaches using universal primers (e.g., rbcl and trnL) have been successful in evaluating polyphagous pest diet, this method is relatively costly and time-consuming. Hence, there is a need for a rapid, specific, sensitive, and cost-effective method to screen for crops in the gut of pests. This is the first study to develop plant-specific primers that target various regions of their genomes, designed using a whole plant genome sequence. We selected Verticillium wilt disease resistance protein (VE-1) and pathogenesis related protein-coding genes 1–5 (PR-1-5) as our targets and designed species-specific primers for 14 important crops in the agroecosystems. Using amplicon sizes ranging from 115 to 407 bp, we developed two multiplex primer mixes that can separate nine and five plant species per PCR reaction, respectively. These two designed primer mixes provide a rapid, sensitive and specific route for polyphagous pest dietary evaluation in agroecosystems. This work will enable future research to rapidly expand our knowledge on the diet preference and range of crops that pests consume in various agroecosystems, which will help in the redesign and development of new crop rotation regimes to minimize polyphagous pest pressure and damage on crops.

A multiplex polymerase chain reaction for the simultaneous detection of the virus and satellite components associated with cotton leaf curl begomovirus disease complex

Cotton leaf curl disease (CLCuD) is caused by a complex of several whiteflies (Bemisia tabaci Genn.)-transmitted begomovirus species, Cotton leaf curl Multan virus (CLCuMuV), Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Alabad virus (CLCuAlV) by individual of mixed infection, associated with Cotton leaf curl Multan betasatellite (CLCuMB) and several alphasatellites. The disease causes major economic losses in cotton in the Indian subcontinent. For monitoring of epidemiology and development of management strategies of CLCuD, a quick, sensitive and effective method capable of detecting all the begomovirus, betasatellite and alphasatellite components associated with CLCuD is required. With this objective, a multiplex polymerase chain reaction (mPCR) assay was developed for the simultaneous detection of these three viral components associated with CLCuD of cotton. Primers for each component were designed based on the retrieved reference sequences from the GenBank. Each pair of primers, designed for each of the respective component, was evaluated for its sensitivity and specificity in both the component-specific simplex polymerase chain reaction (sPCR) and mPCR assay. This report identified three viral component-specific pairs of primers which, in all combinations, amplified simultaneously the CP gene (780 nts) of the begomovirus, the βC1gene (375 nts) of the betasatellite and the Rep gene (452 nts) of the alphasatellite associated with CLCuD in the mPCR assays. The amplified products specific to each component produced by these assays were identified based on their amplicon sizes, and the identities of the viral components amplified were confirmed by cloning and sequencing the amplicons obtained in the mPCR. The mPCR assay was validated using naturally CLCuD-affected cotton plants of the fields. This assay will be useful for rapid detection of CLCuD-associated begomovirus, betasatellite and alphasatellite DNA in field samples, extensive resistance screening in resistance breeding programme, and also monitoring epidemiology for detection of virus and its components when symptoms are mild or absent in the plant.

Identification and Characterization of Nine Novel X-Chromosomal Short Tandem Repeats on Xp21.1, Xq21.31, and Xq23 Regions.

The application of X-chromosomal short tandem repeats (X-STRs) has been recognized as a powerful tool in complex kinship testing. To support further development of X-STR analysis in forensic use, we identified nine novel X-STRs, which could be clustered into three linkage groups on Xp21.1, Xq21.31, and Xq23. A multiplex PCR system was built based on the electrophoresis. A total of 198 unrelated Shanghai Han samples along with 168 samples from 43 families was collected to investigate the genetic polymorphism and forensic parameters of the nine loci. Allele numbers ranged from 5 to 12, and amplicon sizes ranged from 146 to 477 bp. The multiplex showed high values for the combined power of discrimination (0.99997977 in males and 0.99999999 in females) and combined mean exclusion chances (0.99997918 and 0.99997821 in trios, 0.99984939 in duos, and 0.99984200 in deficiency cases). The linkage between all pairs of loci was estimated via Kosambi mapping function and linkage disequilibrium test, and further investigated through the family study. The data from 43 families strongly demonstrated an independent transmission between LGs and a tight linkage among loci within the same LG. All these results support that the newly described X-STRs and the multiplex system are highly promising for further forensic use.

Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a ‘perfect’ agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.

Molecular characterization and phylogenetic analysis of collected mosquitoes (Diptera: Culicidae) from Northcentral Nigeria using mitochondrial COI and ribosomal IGS gene regions

BackgroundMosquitoes are important vectors of disease-causing organisms such as filarial worms, malaria parasites, and arboviruses endemic to sub-Saharan Africa including Nigeria. Malaria is a disease caused by a plasmodium parasite, transmitted by the bite of infected mosquitoes and is no doubt a public health concern. There is limited information on the genetic diversity of mosquitoes in Nigeria. This is necessary because information about the genetic diversity of mosquitoes is a very important step towards vector control and management with the aim to mitigate or eliminate burden resulting from malaria and other diseases caused by mosquitoes. In the present study, we investigated the genetic variability and relatedness of mosquitoes based on the DNA sequences of the mitochondrial cytochrome oxidase subunit I (COI) and ribosomal intergenic non-coding spacer gene regions (IGS). Mosquitoes were collected from five different states in Northcentral Nigeria, they were morphologically identified using standard keys and genomic DNA was extracted. The specific regions of interests were amplified, and the PCR products were then sequenced.ResultsPCR was able to successfully amplify the expected amplicon sizes of COI and IGS sequences (710 and 169) base pairs, respectively. For COI sequence, pairwise genetic distance between mosquito species ranged from 0.00 to 0.17 in the COI sequences. The pairwise genetic distance among Culex, Aedes and Anopheles species in the IGS sequences ranged from 0.000 to 0.118. Phylogenetic analysis of the sequences of the mitochondrial cytochrome oxidase subunit showed that there was genetic diversity amongst the different mosquito species sampled. It effectively showed marked differences between Culicine and Anopheline mosquitoes.ConclusionsThe ribosomal IGS primers used for this study only amplified Anopheles spp. However, it revealed that there is diversity among the Anopheles gambiae and Anopheles arabiensis samples collected. This study concludes that the mitochondrial COI and ribosomal IGS gene regions are reliable markers for mosquito genetic diversity study and will surely yield a reliable result for molecular diversity assessment of mosquito species.

Development and validation of a novel 133-plex forensic STR panel (52 STRs and 81 Y-STRs) using single-end 400bp massive parallel sequencing.

Short tandem repeats (STRs) are the preferred genetic markers in forensic DNA analysis, routinely measured by capillary electrophoresis (CE) method based on the fragment length features. While, the massive parallel sequencing (MPS) technology could simultaneously target a large number of intriguing forensic STRs, bypassing the intrinsic limitations of amplicon size separation and accessible fluorophores in CE, which is efficient and promising for enabling the identification of forensic biological evidence. Here, we developed a novel MPS-based Forensic Analysis System Multiplecues SetB Kit of 133-plex forensic STR markers (52 STRs and 81 Y-STRs) and one Y-InDel (M175) based on multiplex PCR and single-end 400bp sequencing strategy. This panel was subjected to developmental validation studies according to the SWGDAM Validation Guidelines. Approximately 2185 MPS-based reactions using 6 human DNA standards and 8 male donors were conducted for substrate studies (filter paper, gauze, cotton swab, four different types of FTA cards, peripheral venous blood, saliva, and exfoliated cells), sensitivity studies (from 2ng down to 0.0625ng), mixture studies (two-person DNA mixtures), PCR inhibitor studies (seven commonly encountered PCR inhibitors), species specificity studies (11 non-human species), and repeatability studies. Results of concordance studies (413 Han males and 6 human DNA standards) generated by STRait Razor and in-house Python scripts indicated 99.98% concordance rate in STR calling relative to CE for STRs between 41,900 genotypes at 100 STR markers. Moreover, the limitations of present studies, the nomenclature rules and forensic MPS applications were also described. In conclusion, the validation studies based on ~ 2200 MPS-based and ~ 2500 CE-based DNA profiles demonstrated that the novel MPS-based panel meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities, and the STR nomenclature rules should be further regulated to integrate the inconformity between MPS-based and CE-based methods.

Comprehensive Analysis of the Tumoral and Microenvironmental Transcriptome of Lymph Nodes and Bone Marrow in Mantle Cell Lymphoma

The Reverse Transcription Multiplex Ligation-dependent Probe Amplification (RT-MLPA) technique is a method allowing for the semi-quantitative concomitant analysis of a number of transcripts of interest in cells or tissues. It relies on the amplification of complementary DNA (cDNA) derived from messenger ribonucleic acid (mRNA) after binding and ligation of specific probes on adjacent regions. It is thus applicable to the relatively fragmented low-quality RNA that can be derived from formalin-fixed paraffin embedded samples (FFPE), ill adapted to other methods such as RNASeq. Adequate tagging provides specific amplicon sizes for each marker, thus becoming readily identifiable after capillary electrophoresis. Signal normalization further allows to compare relative levels of transcripts in a given run.Here we report on the application of this method to explore the tumoral and microenvironmental, i.e. intrinsic and extrinsic, transcriptome of mantle cell lymphoma (MCL). Data were obtained from i)bone marrow (BM) and lymph nodes (LN) from normal individuals, ii)BM and LN from MCL patients at diagnosis (n=33) and iii)BM and LN from relapsing MCL patients (n=19). Samples were either frozen BM and frozen of FFPE LN. mRNAs were extracted and reverse-transcripted in cDNA before being processed for MLPA investigating for 17 transcripts of interest. The latter explored the intrinsic tumoral expression of CCND1, SOX11 and MKI67. The extrinsic parameters of the microenvironment investigated concerned the monocyte/macrophage compartment (CD14, CD163), T-cells (CD3E/CD3, CD8A/CD8), NK cells (CD94), the costimulation molecule CD40-ligand (CD40LG/CD40L) and immune-checkpoint molecules (CD152/CTLA4, PDC1/PD1, CD274/PDL1, PDCD1LG2/PDL2, INDO1/IDO) as well as cytokines (CSF1/MCSF , IL10, TGFB1).The samples studied were 39 MCL LN, 20 MCL BM, 10 non-tumoral LN and 9 normal BM.RT-MLPA readily differentiated MCL samples from non-tumoral samples by showing high levels of CCND1 and SOX11 transcripts in patients. Extrinsic markers expression, analyzed in principal component analysis (PCA) clearly segregated diseased and normal LN, while the difference was less clear in BM. Macrophage markers transcript levels were lower in patients' LN which also showed a down regulation of global T- and NK-cells yet increased levels of CD8 transcripts. PDL1 expression was higher in MCL BM compared to controls.LN from MCL patients with aggressive characteristics displayed lower CCND1 but higher MKI67 transcripts than other MCL patients' samples. These aggressive forms were found to be significantly associated with high CD14 transcripts in LN and high CD163 in BM. In addition, blastoid morphology was significantly associated with high CD14 (p=.005) and CD163 transcript levels in LN. Regarding T-cells, MCL aggressive forms had significantly lower amounts of CD3E transcripts, yet increased CD8 expression.LN from patients with a high proliferative index displayed higher levels of CD8 but also of CTLA-4, PD-1 and PDL1, suggesting that these T-cells are exhausted.A significant upregulation of the levels of CCND1 transcripts was observed in relapse LN. Conversely, CD163, CD3E, CD40L and CD94 levels were lower in relapsed samples compared to diagnosis, which could reflect immune depletion secondary to immuno-chemotherapy.This study confirms, in an original transcriptomic approach, the great heterogeneity of the MCL microenvironment. Besides being a useful tool at diagnosis, RT-MLPA successfully allowed for a concomitant approach of a large number of transcripts and thus a precise evaluation of intrinsic prognostic and extrinsic features of MCL. Important, yet seldom described, modulations of T-cells, NK cells, macrophages and cytokines were observed, with different profiles segregating MCL with aggressive morphology and those with resistance to treatment. DisclosuresNo relevant conflicts of interest to declare.

A Multiplex PCR Assay Combined with Capillary Electrophoresis for the Simultaneous Identification of Atlantic Cod, Pacific Cod, Blue Whiting, Haddock, and Alaska Pollock.

With an increased consumption of seafood products, food fraud with fish resources has been continuously reported. In particular, codfish has been exploited worldwide as a processed product in fresh, frozen, smoked, canned, or ready-to-eat dish forms. However, it is challenging to identify processed fish products after processing because of their similar morphological characteristics. Substitution and mislabeling of codfish among different species are also happening deliberately or unintentionally. Thus, it is necessary to distinguish cod species to prevent fish adulteration and food fraud. In this study, we developed a multiplex PCR for simultaneously identifying five cod species within Gadidae using capillary electrophoresis. Then, their species-specific primer sets were designed by targeting the mitochondrial cytochrome b gene. Subsequently, the amplicon sizes obtained were 237 bp, 204 bp, 164 bp, 138 bp, and 98 bp for Atlantic cod, Pacific cod, blue whiting, haddock, and Alaska pollock, respectively. The specificity of each primer was further tested using 19 fish species, and no cross-reactivity was observed. The limit of detection of this multiplex PCR assay was 1 pg. The developed multiplex PCR assay can be applied to 40 commercial food products successfully. This detection method will be efficient for managing seafood authentication by simultaneously analyzing multiple cod species.

Simultaneous detection and differentiation between Mycobacterium leprae and Mycobacterium lepromatosis using novel polymerase chain reaction primers.

We have developed a polymerase chain reaction-based method to detect and distinguish Mycobacterium leprae and Mycobacterium lepromatosis using a single set of primers based on a 45-bp difference in the amplicon size of their rpoT gene. This method can also help in detecting the cases of co-infection in a single experiment.

Detection of Genetic markers associated with Plasmodium falciparum drug resistance among malaria patients in Kaduna State, Nigeria

Malaria is a disease of public health concern in Nigeria and sub-Saharan Africa. It is caused by intracellular parasites of the genus Plasmodium. The aim of this study was to detect genetic markers associated with Plasmodium falciparum drug resistance among malaria patients in Kaduna State, Nigeria. The study was a cross-sectional study that lasted from May 2018 to October 2018. Three hundred blood samples were collected from consenting individuals attending selected hospitals, in the three senatorial districts of Kaduna State, Nigeria. Structured questionnaire were used to obtain relevant data from study participants. The blood samples were screened for malaria parasites using microscopy and rapid diagnostic test kit. Polymerase Chain Reaction was used for detection of the drug resistance genes. Pfcrt, pfmdr1, pfdhfr, pfdhps and pfatpase6 genes were detected at expected amplicon sizes from the malaria positive samples. The pfatpase6 PCR amplicons were sequenced and a phylogenetic tree was created to determine their relatedness. Result showed that Pfcrt (80%) had the highest prevalence, followed by pfdhfr (60%), pfmdr1 (36%) and pfdhps (8%). Pfatpase6 was also detected in 73.3% of the samples, and a phylogenetic tree showed relatedness between the pfatpase6 sequences in this study and those deposited in the GenBank. In conclusion, the study detected that Plasmodium falciparum genes were associated with drug resistance to commonly used antimalarials.

Rapid and Sensitive Pathogen Detection by DNA Amplification Using Janus Particle-Enabled Rotational Diffusometry

Rapid and sensitive detection of infectious bacteria is in all-time high demand to prevent the further spread of the infection and allow early medical intervention. In this study, we use rotational diffusometry (RD), a natural phenomenon characterized by Janus particles, to detect pathogens like Escherichia coli by performing amplification of specific genes. This biosensing method is used to measure the change in viscosity of the fluid in the presence and absence of DNA in the solution by capturing images of modified microbeads at 10 Hz by a CCD camera followed by cross-correlation algorithm analysis. Using rotational diffusometry, we have achieved E. coli detection with 50 pg/μL DNA with a measurement time of 30 s and a sample volume of 2 μL. This sensitivity was achieved with 30 thermal cycles for three different amplicons, viz., 84, 147, and 246 bp. Meanwhile, in the case of 10 and 20 thermal cycles, the detection sensitivity was achieved with 0.1 and 1 ng/μL DNA concentrations for a 246 bp amplicon. Compared with conventional PCR, this technique appears to improve the detection time, thereby reaching a turnaround time of less than 60 min. Other studies showed a successful identification of DNA amplification up to 10 thermal cycles with different sizes of amplicons. The effect of DNA concentration, amplicon size, and the number of thermal cycles on the detection of E. coli was examined in detail and represented in the form of three maps. These maps show the clear difference and the advantages of RD method in comparison with conventional PCR. This unconventional and rapid biosensing method can be used further for downstream application of nucleic acid amplification-based pathogen detection and early disease control.

A new host record for Candidatus Phytoplasma cynodontis (16Sr XIV-A) associated with phyllody and fasciation of linseed (Linum usitatissimum) from India.

Linseed commonly called as flaxseed (Linum usitatissimum Linn.) is an important oilseed crop cultivated widely in Northern parts of Karnataka. During, 2019 (January-February), a characteristic disease was noticed with symptoms that resembled phytoplasma or like disease symptoms. The incidence was ranged from 6·5 to 16·5% in the experimental station of Raichur Agricultural University. The typical symptoms observed were virescence of floral parts, fasciation of the inflorescence axis, phyllody, stunted and flattened stem with reduced leaves. Symptomatic and healthy samples were collected and processed for molecular detection of phytoplasma. Total DNA was isolated from four infected plants and two healthy plants. The 16S rDNA region was amplified using P1/P7 followed by R16F2n/R16R2 primer pair which showed the amplification of expected amplicon size from all four infected samples. Furthermore, the SecA gene was amplified using SecA1/SecA3 primers. The PCR amplified products were subjected for direct sequencing from both directions and the consensus sequences were obtained and nBLAST search analysis revealed that the 16Sr RNA and SecA sequences were sharing maximum similarity (100%) with the reference sequence of Ca. P. cynodontis. The sequences were analysed phylogenetically by constructing a Phylogram independently by NJ method along with reference sequence of 16S rRNA region and SecA region retrieved from GenBank database showed that the phytoplasma sequence from linseed phyllody of the present study placed in a distinct clade along with reference sequence of "Ca. P. cynodontis" thus confirming the identity phylogenetically. Furthermore, iPhyClassifier and virtual RFLP proved that the phytoplasma belonged to 16SrXIV (subgroup A) phytoplasma. Previously linseed is known to be associated with 16SrII-D phytoplasma but the association of the 16SrXIV-A group of phytoplasma is not reported so far. Therefore, this is the new host record for Ca. P. cynodontis (16SrXIV-A) phytoplasma associated with linseed stem fasciation, phyllody from India.

Development of Ogura CMS lines in Indian cauliflower and their characterization using agro-morphological traits and mtDNA markers

Cytoplasmic male sterility (CMS) system is most important genetic mechanism for affordable and genetically homogenous hybrid seed production. The present study was aimed to evaluate the CMS lines and their maintainers for floral traits as well as assess suitability of mitochondrial DNA (mtDNA) markers to distinguish the fertile/sterile plants at seedling stage. Morphological observations revealed that introgression of CMS trait significantly reduced flower size (length, diameter), petal size (length, width), stigma and anther length. Out of five mtDNA markers, namely Orf138, orf222-orf224, atp6-orf224, Orf138 and BnTR4, two markers (Orf138 and orf222-orf224) exhibited dominant pattern and clearly distinguished CMS lines from their fertile maintainers. However, atp6-orf 224 and BnTR4 showed polymorphic amplicons of different size between them. These new CMS lines have great potential in hybrid breeding and hybrid seed production in Indian cauliflower. The two mtDNA markers (Orf138 and orf222-orf224) will be useful for early detection of Ogura CMS system during introgression programme and for clarification of doubtful seeds.

Serine hydroxymethyltransferase 2 expression promotes tumorigenesis in rhabdomyosarcoma with 12q13-q14 amplification.

The 12q13-q14 chromosomal region is recurrently amplified in 25% of fusion-positive (FP) rhabdomyosarcoma (RMS) cases and is associated with a poor prognosis. To identify amplified oncogenes in FP RMS, we compared the size, gene composition, and expression of 12q13-q14 amplicons in FP RMS with those of other cancer categories (glioblastoma multiforme, lung adenocarcinoma, and liposarcoma) in which 12q13-q14 amplification frequently occurs. We uncovered a 0.2 Mb region that is commonly amplified across these cancers and includes CDK4 and 6 other genes that are overexpressed in amplicon-positive samples. Additionally, we identified a 0.5 Mb segment that is only recurrently amplified in FP RMS and includes 4 genes that are overexpressed in amplicon-positive RMS. Among these genes, only serine hydroxymethyltransferase 2 (SHMT2) was overexpressed at the protein level in an amplicon-positive RMS cell line. SHMT2 knockdown in amplicon-positive RMS cells suppressed growth, transformation, and tumorigenesis, whereas overexpression in amplicon-negative RMS cells promoted these phenotypes. High SHMT2 expression reduced sensitivity of FP RMS cells to SHIN1, a direct SHMT2 inhibitor, but sensitized cells to pemetrexed, an inhibitor of the folate cycle. In conclusion, our study demonstrates that SHMT2 contributes to tumorigenesis in FP RMS and that SHMT2 amplification predicts differential response to drugs targeting this metabolic pathway.

Occurrence of natural infection of tomato by Potato Spindle Tuber Viroid (PSTVd) in India

A field survey was carried out in districts of southern Karnataka to assess the pospiviroid infections on tomato crop. The tomato plants showing disease symptoms such as severe leaf curling, epinasty, chlorosis, purpling and stunted growth that are characteristics of viroid infection were collected along with asymptomatic plants and the infection was analysed by RT-PCR. A sample collected from the Mandya district showed an amplicon size of ~ 361 bp which confirmed PSTVd infection, while no other Pospiviroids were detected. The amplified PCR products were cloned, sequenced and the resultant sequences were deposited in GenBank. The PSTVd sample inoculated tomato plants also showed typical disease symptoms of viroid infection after four weeks of post-inoculation and was re-confirmed through RT-PCR. This is the first report of the occurrence of natural infection of tomato by PSTVd in India.

Incidence of Alternaria Species Associated with Watermelon Leaf Blight in Korea.

Alternaria leaf blight is one of the most common diseases in watermelon worldwide. In Korea, however, the Alternaria species causing the watermelon leaf blight have not been investigated thoroughly. A total of 16 Alternaria isolates was recovered from diseased watermelon leaves with leaf blight symptoms, which were collected from 14 fields in Korea. Analysis of internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RNA polymerase II second largest subunit (RPB2) were not competent to differentiate the Alternaria isolates. On the contrary, analysis of amplicon size of the histone H3 (HIS3) gene successfully differentiated the isolates into three Alternaria subgroups, and further sequence analysis of them identified three Alternaria spp. Alternaria tenuissima, A. gaisen, and A. alternata. Representative Alternaria isolates from three species induced dark brown leaf spot lesions on detached watermelon leaves, indicating that A. tenuissima, A. gaisen, and A. alternata are all causal agents of Alternaria leaf blight. Our results indicate that the Alternaria species associated watermelon leaf blight in Korea is more complex than reported previously. This is the first report regarding the population structure of Alternaria species causing watermelon leaf blight in Korea.

Characterization of Domain V Mutations in Clinical Isolates of Helicobacter pylori in Pakistan and Their Effect on Clarithromycin MIC.

PurposeClarithromycin is commonly prescribed for H. pylori infection. Domain V mutations are responsible for clarithromycin resistance. This study was aimed to characterize the clarithromycin resistance and its associated mutations in clinical isolates of H. pylori in Pakistan.Materials and MethodsInfection was diagnosed in 93 patients’ biopsies using culture, rapid urease test, 16S rRNA, and vacA gene multiplex PCR. Clarithromycin resistance was assessed by the agar dilution method. Mutations were detected by PCR-RFLP using 46 (1.4 kb) domain V fragments. Sequencing was executed for 13 domain V fragments, of which 12 showed unusual amplicon size (1.2 kb) and 01 had a new MboII RFLP pattern.ResultsA total of 48 (83%) strains were obtained from 58 (62.3%) PCR H. pylori-positive samples. Resistance (MIC ≥ 0.001 mg/mL) and intermediate resistance phenotype (MIC = 0.0005 mg/mL) was observed in 22 (46%), and 10 (21%) isolates, respectively. The primary resistance was found in 23 (39.6%) samples. PCR-RFLP detected A2142G, A2143G, and double mutations in 19, 04, and 01 resistant strain, respectively. Sequencing of 10 amplicons obtained from intermediated resistant strains and 03 amplicons from resistant strains showed 138 new mutations. Among them, T2182C was also seen in 04 intermediated resistant isolates, whereas A2142G, A2143G, and A2143C were observed in resistant isolates. The new MboII RFLP pattern in an intermediated resistant strains was due to A1761G mutation.ConclusionH. pylori domain V mutations showed extensive diversity. Multiple mutations in domain V may give endurance to H. pylori against clarithromycin. Further investigations on the molecular mechanism of antibiotic resistance in H. pylori seem crucial at this stage.