First-round PCR Research Articles

First report of a 'Candidatus phytoplasma asteris' related strain (16SrI) associated with phyllody and witches'-broom symptom in Pisum sativum in Hisar, India.

Pea (Pisum sativum L.) is a leguminous vegetable crop, and India holds the fourth position in the production, primarily contributed by three major states: Uttar Pradesh, Madhya Pradesh, and Punjab (Anonymous, 2022). However, a survey conducted in February 2023 at the National Seed Corporation Farm (15 hectares) in Hisar, Haryana, revealed deformities in the growth of some pea plants. Approximately 10% of these plants exhibited a distinct bushy appearance, accompanied by phyllody and witches'-broom symptoms, characterized by deformed leaves and short internodes (Fig. 1). In response to these observed anomalies, a detailed molecular analysis was conducted at the Plant Pathology Laboratory, IARI, New Delhi. The investigation involved the collection of ten samples each from symptomatic and asymptomatic plants, and DNA was extracted from 100 mg leaf midribs using the CTAB method (Ahrens and Seemüller, 1992). The extracted DNA (100 ng/µl) along with one positive (Catharanthus roseus from glass house, 16SrII-D group) and one negative (without template DNA), served as a template for PCR reactions targeting the 16Sr RNA and secA genes. Universal primers P1/P7 and secAfor1/secArev3 were employed in the first round of PCR for the respective genes. Subsequently, the product from the 1st round was diluted and used as a template for the 2nd round of PCR with primers R16F2n/R16R2 for 16Sr RNA and secAfor2/secArev3 for the secA gene (Gundersen and Lee 1996; Deng and Hiruki 1991; Hodgetts et al. 2008). This nested-PCR approach yielded distinct bands, approximately 1.2 kb (16Sr RNA) and 480 bp (secA), from the DNA of all ten symptomatic plants and positive sample, while no bands were observed in any of the asymptomatic plants. The nested PCR products were sequenced by BBS labs (Barcode biosciences, Bengaluru). The 16Sr RNA gene sequences, showing 100% similarity, were submitted to NCBI as representative sequences (Acc. No. OQ690013, OQ690014, OQ709133, OQ709134). Similarly, secA gene sequences were submitted with Acc. Nos. OR604283-86. BLAST analysis revealed a maximum 99.76% identity with Onion yellows phytoplasma and 'Ca. P. asteris' reference strain for 16Sr RNA gene, and a maximum 100% identity with 'Elaeis guineensis' stunt phytoplasma for secA gene. The phylogenetic tree constructed using the 16S rRNA and secA gene sequences indicated that the pea phytoplasma strains of this study clustered with 'Ca. P. asteris' (16SrI-B) related strains (Fig 2a and 2b). Additionally, the 16S rRNA sequences from this study, when subjected to Virtual RFLP using iPhyclassifier (Zhao et al. 2009), exhibited a pattern (Fig. 3) matching the reference pattern of the 16S group I, subgroup B (GenBank accession: M30790), with a similarity coefficient of 1.0. Previously, Rao et al. (2021) reported several crops associated with the 16SrI ribosomal group, including eight sub-groups from India. However, this report represents the first instance of a phytoplasma 16SrI-B group associated with phyllody and witches'-broom symptoms in pea, both in India and globally. Considering the economic importance of pea as a vegetable crop, the observed disease incidence and affected area are significant. Urgent attention is required to conduct additional research and implement preventive measures to avert the potential outbreak of this disease in the near future.

Presence of potentially novel Helicobacter pylori-like organisms in gastric samples from cats and dogs

While seven gastric non-Helicobacter pylori Helicobacter (NHPH) species are known to commonly colonize the stomach of cats and dogs, the potential of H. pylori and H. pylori-like organisms to infect animals remains controversial and was investigated in this study using gastric samples of 20 cats and 27 dogs. A Helicobacter genus-specific 16 S rRNA PCR assay, H. pylori-specific ureAB and glmM PCR assays and a nested PCR detecting 23 S rRNA in a Helicobacter genus-specific manner in a first round of PCR and a H. pylori-specific manner in a second round, were performed in combination with sequencing. Histopathological and anti-Helicobacter immunohistochemical evaluations were also performed. Based on 16 S rRNA sequence analysis, 39/47 animals (83%) appeared infected with canine/feline gastric NHPHs in the corpus and/or antrum. H. pylori-specific ureAB amplicons were obtained in samples of 22 stomachs (47%). One canine antrum sample positive in the ureAB assay was also positive in the H. pylori-specific glmM assay. While 36/47 (77%) animals had a positive sample in the first round of the nested 23 S rRNA PCR assay, all samples were negative in the second round. Sequence analysis of obtained amplicons and immunohistochemistry point towards the presence of unidentified H. pylori-like organisms in cats and dogs. Histopathological examination suggests a low pathogenic significance of the gastric Helicobacter spp. present in these animals. In conclusion, cats and dogs may be (co-)infected with gastric Helicobacter organisms other than the known gastric NHPHs. Culture and isolation should be performed to confirm this hypothesis.

Development of a simplified and cost-effective sample preparation method for genotyping of human papillomavirus by next-generation sequencing.

High-risk human papillomavirus (HPV) infection is the most common cause of cervical cancer, but low-risk HPV strains can sometimes also be involved. Although HPV genotyping techniques used in clinical diagnosis cannot detect low-risk HPV, next-generation sequencing (NGS) can detect both types. However, DNA library preparation is complicated and expensive. The aim of this study was to develop a simplified, cost-effective sample preparation procedure for HPV genotyping based on next-generation sequencing (NGS). After DNA extraction, a first round of PCR was performed using modified MY09/11 primers specific for the L1 region of the HPV genome, followed by a second round of PCR to add the indexes and adaptors. Then, the DNA libraries were purified and quantified, and high-throughput sequencing was performed using an Illumina MiSeq platform. The sequencing reads were compared with reference sequences for HPV genotyping. The limit of detection for HPV amplification was 100 copies/µl. Analysis of the correlation of pathological cytology with the HPV genotype in individual clinical samples showed that HPV66 was the most common genotype found in the normal stage, whereas HPV16 was the main genotype found in low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and cervical cancer. This NGS method can detect and identify several HPV genotypes with 92% accuracy and 100% reproducibility, and it shows potential as a simplified and cost-effective technique for large-scale HPV genotyping in clinical samples.

The Cancer-Related Circrna Cirs-7 (CDR1-AS) Is Differentially Expressed in CD138+ Cells of Patients with Plasma Cell Disorders and Predicts Unfavorable Overall Survival in Multiple Myeloma

Introduction: During the last decades, different non-coding RNAs have been implicated in the biology and prognosis of multiple myeloma (MM). Circular RNAs (circRNAs) constitute a class of non-coding RNAs with a high potential as molecular biomarkers. Recently, ciRS-7 (also known as CDR1-AS), an important circRNA regulating miR-7-5p and other cancer-related miRNAs, was shown to be downregulated in MM patients’ cells exhibiting acquired resistance to immunomodulatory drugs (IMiDs). Our goal was to study the expression of this circRNA in MM, compare the results with those of patients with smoldering myeloma (SMM) and monoclonal gammopathy of undetermined significance (MGUS) and evaluate its prognostic significance. Methods: Bone marrow aspirate (BMA) samples were collected from 177 adult patients with plasma cell disorders. The presence of high-risk cytogenetic abnormalities, namely del(17p), t(4;14), t(14;16), and (+1q), was assessed using fluorescence in situ hybridization (FISH). MM patients were classified according to the ISS and revised ISS (R-ISS) staging systems. Whole-body low-dose computed tomography was used to assess osteolysis. Next, CD138-positive selection of plasma cells among mononuclear cells of BMA samples was performed. Moreover, three human MM cell lines (L-363, H929 and U266) were propagated. Total RNA was extracted from CD138+ enriched BMA samples and the MM cell lines, and reverse-transcribed. A first-round PCR was used to pre-amplify ciRS-7 and GAPDH (i.e. reference gene) transcripts, before their quantification via real-time qPCR. In both reactions for ciRS-7 amplification, divergent PCR primers were used, to achieve high specificity and accuracy. ciRS-7 expression was measured in relative quantification units (RQU). Results: The cohort of patients consisted of 111 newly diagnosed MM, 35 SMM and 31 with MGUS. In total, 107 out of the 177 cases (60.5%) were male and 70 (39.5%) were female. The median age of all patients at diagnosis was 70 years (range: 35-93 years). According to the R-ISS staging system, 24 (21.6%) MM patients had R-ISS stage I, 62 (55.9%) R-ISS stage II and 25 (22.5%) had R-ISS stage III. Seventy-seven (69.4%) MM patients presented with osteolytic disease at diagnosis. Regarding front-line therapy of MM patients, 12/111 (10.8%) received proteasome inhibitor (PI)-based therapy, 7 (6.3%) received IMiD-based treatment, 74 (66.7%) were treated with combinations of PI and IMiD, and 18 (16.2%) received anti-CD38-based treatment. The intracellular levels of the ciRS-7 circRNA were different between MM, SMM and MGUS patients’ CD138+ plasma cells (median values were: for MM patients 0.40 RQU vs. 1.74 RQU for SMM patients vs. 2.79 RQU for MGUS patients; P<0.001). Moreover, ciRS-7 levels in U266 and H929 cells were much lower (2,165- and 26,616-fold) than in L-363 cells. ROC analysis showed that ciRS-7 expression can effectively discriminate between MM and SMM patients (AUC=0.65, 95% CI=0.56-0.75, P=0.007). ciRS-7 levels did not significantly differ among MM patients with different ISS or R-ISS stages and were not associated with any assessed cytogenetic abnormality or the presence of osteolytic lesions. Regarding the progression-free (PFS) and overall survival (OS), out of 106 MM patients with survival data, patients with high levels of ciRS-7 have lower survival probabilities. As shown in Figure 1, the median PFS of MM patients with high ciRS-7 (high: defined as higher of the median value) was 19 months vs. 25 months for patients with low ciRS-7 (lower values than the median) (P=0.046); similarly, the mean OS of ciRS-7 (high) MM patients was 25 months vs. 30.5 months for ciRS-7 (low) MM patients (P=0.008). Bootstrap multivariate Cox regression analysis showed that the unfavorable prognostic value of ciRS-7 expression with regard to OS is independent of R-ISS stage and age of MM patients (HR=2.6, BCa 95% CI=0.91-4.75, bootstrap P=0.041). Conclusions: Expression levels of ciRS-7, a cancer-related circRNA, in CD138+ plasma cells are significantly different among patients with MM, SMM and MGUS. Most likely, high ciRS-7 levels predict an unfavorable prognosis in MM, independently of the R-ISS stage and age. Combining our results with the limited existing literature providing evidence for ciRS-7 implication in response to IMiD therapy, its biological role in MM deserves further elucidation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular identification of entamoeba Dispar and entamoeba Moshkovskii by using nested multiplex PCR in symptomatic indivisuals

The current study was for determine Entamoeba dispar &amp; Entamoeba moshkovskii in symptomatic individuates by molecular identification ( nested multiplex PCR ) in order obtained for an accurate diagnosis of these Entamoeba species. Total stool samples (120) that collected from symptomatic patients with diarrhea. These samples were collected from patients according to age, residency during the period from ( the first of November 2021 to the end of January 2022). All samples undergo for a direct microscopic examination, The results revealed that 100(73.18%)were positive samples with Entamoeba spp. The results of microscopic examination recorded high infection rate in age group in (4-10)years83.33%. In contrast, recorded high prevalence in rural areas 76.31% than urban areas 73.68% . Showed the method nested multiplex polymerase chain reaction by using 18s rRNA small subunit gene, the first round of PCR showed the infection rate by Entamoeba spp 87(65.41%) out of 120 stool samples .The second round of nested multiplex PCR for these positive samples by using two specific primers revealed mix infection of Entamoeba dispar &amp; Entamoeba moshkovskii with Entamoeba histolytica 5(6.25%), 2(2.5%) respectively, with significant difference at P&lt;0.05.

First report of a 'Candidatus Phytoplasma aurantifolia'-related strain (16SrII-V) associated with phyllody, virescence, and shoot proliferation of sweet William (Dianthus barbatus) in Taiwan.

The sweet William (Dianthus barbatus) is an ornamental belonging to the Caryophyllaceae family; the species produces clusters of flowers that comes in various colors and is grown commonly as garden plants (Lim 2014). In February 2021, sweet Williams showing symptoms typical of phytoplasma diseases were found in a garden located in Wufeng District, Taichung, Taiwan (24°04'37.6"N 120°43'20.4"E). Infected plants exhibited virescence and phyllody symptoms and produced an abnormal number of new shoots from the base of the flowers/flower-like structures (Figure S1) as well as the base of the plants. Among the fifteen plants grown in the area, two exhibited such symptoms. The two symptomatic plants, along with five symptomless plants were sampled. Two flower-like structures were collected from each of the symptomatic plants, and two flower samples were collected for each symptomless plant (Figure S2). Total DNA were extracted from each sample using the Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics) and subjected to diagnostic PCR using primers P1/P7 (Schneider et al. 1995). All four symptomatic samples produced a 1.8-kb amplicon and the ten symptomless samples did not. The amplification products were diluted fifty-fold and used in a second round of PCR using primers R16F2n/R16R2 (Gundersen and Lee 1996). Again, only the symptomatic samples produced an expected 1.25-kb amplicon. A sample was selected for each plant and the PCR products from the first round of PCR were cloned using the pGEM-T Easy Vector System (Promega Inc.) and sequenced (three clones per sample). Fragments of the 16S rRNA gene (1,248 bp; GenBank accession: MW788688) were analyzed using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi). Sequences obtained from the two infected plants were identical, and were classified to the 16SrII-V subgroup with similarity coefficients of 1.0; they also shared 98.6% similarity with the sequence of a 'Candidatus Phytoplasma aurantifolia' reference strain (accession: U15442). BLASTn results indicated that the 16S rRNA gene sequences detected were identical to those of 16SrII-V phytoplasmas affecting mungbean (accession: MW319764), lilac tasselflower (accession: MT420682), peanut (accession: JX403944) and green manure soybean (accession: MW393690) found in Taiwan. To corroborate the above results, 16SrII group-specific primers were used to conduct nested and semi-nested PCR targeting the pathogen's 16S rRNA gene (outer primers: rpF1C/rp(I)R1A; inner primers: rp(II)F1/rp(II)R1; Martini et al. 2007) and immunodominant membrane protein gene (imp; outer primers: IMP-II-F1/IMP-II-R1; inner primers: IMP-II-F2/IMP-II-R1; Al-Subhi et al. 2017). In both assays, the symptomatic samples produced the expected amplicons and the symptomless samples did not. The coding sequence of the imp gene (519 bp; accession: MW755353) was the same among all symptomatic samples, and shared 100% identity with that of the peanut witches'-broom phytoplasma (16SrII; accession: GU214176). To our knowledge, this is the first report of a 16SrII-V phytoplasma infecting sweet Williams in Taiwan. Since 16SrII-V phytoplasmas have also been found infecting mungbeans and peanuts in Taiwan (Liu et al. 2015), the findings here suggest that by serving as a natural host in the field, the sweet William may potentially contribute to the spread of 16SrII-V phytoplasmas to food crops.

Maize (Zea mays L.): A New Host for Ligustrum witches' Broom Phytoplasma.

In the 2019–2020 growing season, two corn fields located in İmamoğlu town (Adana Province, Turkey) were surveyed following the appearance of phytoplasma-like symptoms on maize plants. A total of 40 samples were collected and tested in first-round and nested PCR using universal primer pairs P1/P7 and R16F2n/R16R2, respectively. All maize-diseased plants reacted positively, whilst no PCR amplifications were obtained from asymptomatic plants. Blast sequence analysis of R16F2n/R16R2-primed amplicons from different maize isolates showed 99.2% to 100% of identity with the 16S rRNA gene of Ligustrum witches’ broom phytoplasma (LiWBP). To gain additional molecular information on the 16S ribosomal RNA and 23S rRNA intergenic spacer region of LiWBP, not identified previously, the P1/P7-primed amplicons were also sequenced and analyzed. The results show that maize isolates from Turkey share 99.6% to 100% of identity among them, whereas the highest identity found (91%) was with members of groups 16SrII and 16SrXXV (peanut and tea witches’ broom groups, respectively). This distant relationship between LiWBP and members of 16SrII and XXV was also confirmed by RFLP and phylogenetic analyses. This is the first finding of LiWBP on maize in nature, where it was found responsible for phyllody disease of corn plants in Turkey. The additional molecular information acquired in this study on the 16S–23S rRNA intergenic spacer region of LiWBP further corroborates its distant relationship to any other phytoplasma groups.

Studying KLF6 and PEPD‐gene Promoter by Chromatin Immunoprecipitation Assay

Prolidase (PEPD) is a ubiquitously expressed cytosolic dipeptidase that has the unique ability to cleave proline or hydroxyproline on the C‐terminus of imidodipeptides and imidotripeptides. The reaction is vital for collagen degradation. Since collagen is the most abundant protein in the body, our objective is to get a better understanding of the enzymes involved in its turnover is crucial. In silico analysis on prolidase promoter showed that putative transcription factor of interest is KLF6. To test the hypothesis that the expression of PEPD gene may be regulated by the binding of the transcription factor KLF6 to candidate binding sites we used chromatin immunoprecipitation (ChIP) assay. HEK293T cells were transfected with FLAG‐hKLF6 plasmid and the cellular lysates were obtained to measure the KLF6 protein expression by immunoblotting. After that, purified DNA from the ChIP assays were used in end‐point PCR with primers targeting candidate KLF6‐binding sites in PEPD promoter. The primers used were expected to yield a 615 bp amplicon. PCR products were resolved on 1% agarose/TAE gel. The second round of PCR was performed in which endpoint PCR products from the first round of PCR were used as a template for nested PCR with primers designed to amplify 164 bp. Nevertheless, our results showed no detectable PEPD promoter‐specific signals in the PCR analysis of the ChIP products. However, due to the inherent caveats in the experimental design, choice of reagents, and analysis method, it would be premature to conclude at this time point that PEPD expression is not regulated by KLF6. Accordingly, the design of future experiments will effectively address the limitations of this current work and will help reveal the identity of transcription factors regulating PEPD expression.Support or Funding InformationSupported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐Aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).

High prevalence of norovirus and rotavirus co-infection in children with acute gastroenteritis hospitalised in Brazzaville, Republic of Congo.

To evaluate the clinical severity of diarrhoea associated to viral co-infection in children with acute gastroenteritis. About 461 children under five years hospitalised with acute diarrhoea (266 males and 187 females) were enrolled in the study. Using stool samples, rotavirus and adenovirus infections were investigated by ELISA, and norovirus infections by nested duplex RT-PCR. We assessed social, demographic, clinical and behavioural conditions that might influence the occurrence of rotavirus, adenovirus and norovirus infections. Mono-viral infection was detected in 49% and mixed viral infection in 12% of patients. The prevalence of mixed infection was neither dependent on age nor sex. Three samples were infected with all three viruses. A significant association was found between fever (axillary temperature>37.5°C) and rotavirus-norovirus dual infection (aOR (CI 95%) = 2.1 (1.14-3.84), P=0.016; aOR (CI 95%) = 0.37 (0.19-0.73), P=0.004). Mixed infection was the most common during the dry season from June to October (71.4% versus 54.7%, P=0.023). Co-infection with both rotavirus and norovirus is common in under-five hospitalised children but does not contribute to the severity of the disease.

A sensitive nested multiplex RT-PCR assay for the simultaneous detection of three common viruses infecting pear plants

A highly sensitive nested multiplex reverse transcription-polymerase chain reaction (nmRT-PCR) assay was developed for the simultaneous detection of Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV) infecting pear trees. In the assay, a set of three forward primers specific to each of the three viruses and a universal reverse primer was used as external primers in the first-round PCR, which was followed by a second-round PCR developed previously. The nmRT-PCR assay was 104 times more sensitive than conventional mRT-PCR assay in detecting the three viruses in in vitro pear plantlets. This assay was subsequently used to detect these viruses in leaf and bark samples of cultivated and wild pear trees from orchards and demonstrated to be highly sensitive and reliable. This is the first report describing a use of nmRT-PCR for the sensitive and simultaneous detection of the three viruses infecting pear plants. The assay would be useful for the certification of pear planting materials and surveillance of nursery stocks.

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

IPair and iPair+: cognate pairing of human TCR alpha and beta V-regions and phenotypic analysis of single cells using NGS.

Abstract Immune repertoire amplification of T and B cell receptors coupled to NGS provides detailed, sequence-level insight into the immune system. However, information related to the cognate pairing of alpha and beta chains is lost once RNA extraction is performed on the entire sample (or bulk sample). Here, we describe a very sensitive method that allows for the amplification of both human TCR alpha and beta chains including additional phenotypic markers from single cells in the same reaction well. We have reduced the RT-PCR chemistry as low as 1.5 μL, thus providing a cost-effective method to analyze single cells from standard FACs instruments. Plate processing procedures are also simplified since RT-PCR of all targets can be performed in the same plate. Under optimal conditions, the amplification success rate is greater than 90%, with successfully identified pairs as high as 75% of those amplified. During the first round of PCR, RT-PCR is performed with nested, multiplex primers covering both the alpha and beta locus (iPair) and 30 phenotypic markers (iPair+). Communal forward and reverse binding sites are included on the 5′ end of the inside primers. Included on the reverse primer is an in-line 6 nt barcode, which serves as a plate identifier so that multiple 96-well plates may be multiplexed in the same sequencing flow cell. After RT-PCR1, the first round PCR1 products are rescued using beads. A second PCR is performed with dual-indexed primers that complete the sequencing adaptors introduced during PCR1 and provide plate positional information for the sequenced products. An analysis tool has also been developed to help visualize pairing across a plate and to identify those pairs that would be likely candidates for further investigation.

First Report of a 16SrXIV Group Phytoplasma Associated With Witches’-Broom Disease of Acid Lime (Citrus aurantifolia) in India

HomePlant DiseaseVol. 101, No. 5First Report of a 16SrXIV Group Phytoplasma Associated With Witches’-Broom Disease of Acid Lime (Citrus aurantifolia) in India PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of a 16SrXIV Group Phytoplasma Associated With Witches’-Broom Disease of Acid Lime (Citrus aurantifolia) in IndiaD. K. Ghosh, S. Bhose, P. Sharma, A. Warghane, M. Motghare, M. S. Ladaniya, M. K. Reddy, V. Thorat, and A. YadavD. K. GhoshSearch for more papers by this author, S. BhoseSearch for more papers by this author, P. SharmaSearch for more papers by this author, A. WarghaneSearch for more papers by this author, M. MotghareSearch for more papers by this author, M. S. LadaniyaSearch for more papers by this author, M. K. ReddySearch for more papers by this author, V. ThoratSearch for more papers by this author, and A. YadavSearch for more papers by this authorAffiliationsAuthors and Affiliations D. K. Ghosh S. Bhose P. Sharma A. Warghane M. Motghare M. S. Ladaniya , ICAR-Central Citrus Research Institute, Nagpur-440 033, India M. K. Reddy , ICAR-Indian Institute of Horticultural Research, Bangalore-560 089, India V. Thorat A. Yadav , Microbial Culture Collection, National Center for Cell Science, Pune - 411 021, India. Published Online:9 Mar 2017https://doi.org/10.1094/PDIS-11-16-1549-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Acid lime [Citrus aurantifolia (L.) Swingle] is an important citrus cultivar that constitutes nearly 20% of total citrus production in India. During a survey conducted in April 2014 to February 2015, conspicuous symptoms of small chlorotic leaves with highly proliferated shoots and shortened internodes resembling witches’-broom disease of acid lime (WBDL) were observed in four out of 22 acid lime groves in Nagpur district of Maharashtra state, India. Seven representative field samples from symptomatic plants were graft-inoculated to healthy acid lime seedlings under greenhouse conditions and all developed characteristic symptoms 18 to 20 months after grafting. Numerous phytoplasma-like bodies of 100 to 800 nm in diameter and bounded by poorly defined membrane were observed in sieve tubes of diseased samples but not in similar sections from healthy leaves when ultrathin sections of leaf midrib were observed under a JEM 100S transmission electron microscope. PCR was used to confirm the presence of phytoplasma in infected tissues from 20 symptomatic and five asymptomatic plants. Samples were analyzed using P1/P7 primer pairs for the first round of PCR and the products were subsequently amplified with nested primer pairs R16F2n/R16R2 (Lee et al. 2004). An expected amplicon of ∼1.2 kb was obtained from 18 diseased plant tissues while it was not observed in asymptomatic plants. Four amplicons obtained from different field samples using R16F2n/R16R2 were purified, sequenced, and the resultant sequences were deposited in GenBank (KY024345, KY024346, KY024347, KY024348). All obtained sequences were 100% identical. The results of NCBI-BLAST analysis further revealed that the 16S rRNA gene sequences shared 99.9% similarity with that of the ‘Candidatus Phytoplasma cynodontis’ strains (LT558777, KF234570, AB741630). Phylogenetic analysis of obtained sequences showed that the WBDL phytopalsma formed a distinct clade with ‘Ca. Phytoplasma cynodontis’ strain BGWL-C1. In silico RFLP analysis of the aligned 16S rRNA gene sequence of the WBDL phytoplasma was performed with 17 distinct restriction enzymes using iPhyClassifier (Zhao et al. 2009) and the results were compared with each phytoplasma group and subgroup. Based on the analysis, WBDL phytoplasma 16S rRNA gene sequences shared 99.8% similarity with that of the ‘Ca. Phytoplasma cynodontis’ reference strain (AJ550984). Moreover, the virtual RFLP pattern derived from the query 16S rRNA gene F2nR2 fragment was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group XIV, subgroup A (AJ550984) (Marcone et al. 2004). Taken together, these results indicate that the phytoplasma associated with WBDL in India is a ‘Ca. Phytoplasma cynodontis’-related strain and belongs to 16SrXIV-A subgroup. To the best of our knowledge, this is the first report of occurrence of 16SrXIV group phytoplasma associated with WBDL not only in India but also in the world. It will be interesting to identify potential plant reservoir and insect vectors of the pathogen in this region. The disease adversely affects fruit yield and has the potential to significantly reduce economic condition of citrus growers of the region.

Application of Stochastic Labeling with Random-Sequence Barcodes for Simultaneous Quantification and Sequencing of Environmental 16S rRNA Genes.

Next-generation sequencing (NGS) is a powerful tool for analyzing environmental DNA and provides the comprehensive molecular view of microbial communities. For obtaining the copy number of particular sequences in the NGS library, however, additional quantitative analysis as quantitative PCR (qPCR) or digital PCR (dPCR) is required. Furthermore, number of sequences in a sequence library does not always reflect the original copy number of a target gene because of biases caused by PCR amplification, making it difficult to convert the proportion of particular sequences in the NGS library to the copy number using the mass of input DNA. To address this issue, we applied stochastic labeling approach with random-tag sequences and developed a NGS-based quantification protocol, which enables simultaneous sequencing and quantification of the targeted DNA. This quantitative sequencing (qSeq) is initiated from single-primer extension (SPE) using a primer with random tag adjacent to the 5’ end of target-specific sequence. During SPE, each DNA molecule is stochastically labeled with the random tag. Subsequently, first-round PCR is conducted, specifically targeting the SPE product, followed by second-round PCR to index for NGS. The number of random tags is only determined during the SPE step and is therefore not affected by the two rounds of PCR that may introduce amplification biases. In the case of 16S rRNA genes, after NGS sequencing and taxonomic classification, the absolute number of target phylotypes 16S rRNA gene can be estimated by Poisson statistics by counting random tags incorporated at the end of sequence. To test the feasibility of this approach, the 16S rRNA gene of Sulfolobus tokodaii was subjected to qSeq, which resulted in accurate quantification of 5.0 × 103 to 5.0 × 104 copies of the 16S rRNA gene. Furthermore, qSeq was applied to mock microbial communities and environmental samples, and the results were comparable to those obtained using digital PCR and relative abundance based on a standard sequence library. We demonstrated that the qSeq protocol proposed here is advantageous for providing less-biased absolute copy numbers of each target DNA with NGS sequencing at one time. By this new experiment scheme in microbial ecology, microbial community compositions can be explored in more quantitative manner, thus expanding our knowledge of microbial ecosystems in natural environments.

PM 7/128 (1) Xanthomonas axonopodis pv. allii

Specific scopeThis Standard describes a diagnostic protocol for Xanthomonas axonopodis pv. allii.This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.Specific approval and amendmentFirst approved in 2016‐09.

FIRST REPORT OF CUCUMBER MOSAIC VIRUS IN ANEMONE sp. IN GREECE

Virus-like symptoms, such as severe stunting and mosaic, were observed in a greenhouse-grown Anemone sp. (cv. Jerusalem), in Pella (northern Greece) in January 2014. Foliar samples were collected from 20 symptomatic and five symptomless plants and subjected to three initial tests: (i) DAS-ELISA to check for the presence of Cucumber mosaic virus (CMV), (ii) RT-PCR using generic primers for the detection of potyviruses (Pappu et al., 1993) and (iii) PCR for phytoplasmas (Gundersen and Lee, 1996). CMV was detected in all symptomatic plants but i n none of the asymptomatic ones, whereas PCR-based tests for potyviruses or phytoplasmas resulted negative. In order to verify the results of serological tests, a nested RT-PCR was performed on total RNA extracts from all 25 samples. Degenerate primers CMVup624a (5′- ATGGACAAATCTGRATC-3′) and CMVdo1244a (5′- TGRTGCTCRAYGTCKACATGA-3′), used in the first round of PCR were followed by the set CMVup624b (5′- GGACAAATCTGRATCTCCCAATGC-3′) and CMVdo1244b (5′- TGCTCRAYGTCRACATGAAG-3′), designed to amplify a 622 nt- long fragment of the viral coat protein gene. A mplicons of t h e expected size were obtained from all the symptomatic samples but not from the asymptomatic ones. Sequencing of two randomly chosen PCR products (GenBank accession Nos LN810056, LN810057) showed 99% nucleotide identity with CMV isolates from various hosts (KC763473, EU191027). Phylogenetic analyses showed that studied CMV isolates from Anemone sp. belong to sub-Group II. Finally, CMV was mechanically transmitted from the original symptomatic plants onto Cucurbita pepo and Nicotiana benthamiana, and its presence was confirmed by DAS-ELISA. To our knowledge this is the first report of CMV in Anemone sp. in Greece.

Nested PCR Biases in Interpreting Microbial Community Structure in 16S rRNA Gene Sequence Datasets.

BackgroundSequencing of the PCR-amplified 16S rRNA gene has become a common approach to microbial community investigations in the fields of human health and environmental sciences. This approach, however, is difficult when the amount of DNA is too low to be amplified by standard PCR. Nested PCR can be employed as it can amplify samples with DNA concentration several-fold lower than standard PCR. However, potential biases with nested PCRs that could affect measurement of community structure have received little attention.ResultsIn this study, we used 17 DNAs extracted from vaginal swabs and 12 DNAs extracted from stool samples to study the influence of nested PCR amplification of the 16S rRNA gene on the estimation of microbial community structure using Illumina MiSeq sequencing. Nested and standard PCR methods were compared on alpha- and beta-diversity metrics and relative abundances of bacterial genera. The effects of number of cycles in the first round of PCR (10 vs. 20) and microbial diversity (relatively low in vagina vs. high in stool) were also investigated. Vaginal swab samples showed no significant difference in alpha diversity or community structure between nested PCR and standard PCR (one round of 40 cycles). Stool samples showed significant differences in alpha diversity (except Shannon’s index) and relative abundance of 13 genera between nested PCR with 20 cycles in the first round and standard PCR (P<0.01), but not between nested PCR with 10 cycles in the first round and standard PCR. Operational taxonomic units (OTUs) that had low relative abundance (sum of relative abundance <0.167) accounted for most of the distortion (>27% of total OTUs in stool).ConclusionsNested PCR introduced bias in estimated diversity and community structure. The bias was more significant for communities with relatively higher diversity and when more cycles were applied in the first round of PCR. We conclude that nested PCR could be used when standard PCR does not work. However, rare taxa detected by nested PCR should be validated by other technologies.

A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method

Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function. We introduced a nested-SOE-PCR (N -SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR. Genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application. In comparison to the conventional SOE-PCR, the improved method (i.e. N-SOE-PCR) increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products. By applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE.

Next Generation Amplicon Sequencing of Immunoglobulin Heavy Chain Gene Rearrangaments for Minimal Residual Disease (MRD) Stratification in Childhood Acute Lymphoblastic Leukemia (ALL): A Comparison with Classical qPCR-Based Technique

▪IntroductionMRD is an important predictor of outcome in childhood ALL. Since 2000, MRD detected by quantitative PCR (qPCR) for immunoglobulin and T-cell receptor gene rearrangements with a minimal sensitivity of 1E-04 has been used for risk group stratification in pediatric BFM trials. Next generation sequencing (NGS) permits rapid parallel sequencing of large numbers of DNA segments. It can overcome most of the limitations of qPCR: it allows highly specific molecular detection of MRD without laborious optimization of patient-specific assays. Moreover it enables not only monitoring of malignant clone but also shows the picture of entire immune background.AimsTo develop an assay for immunoglobulin heavy chain (IgH) rearrangements detection on Ion Torrent PGM/Ion Proton platforms and compare the MRD levels with qPCR at BFM stratification timepoints.MethodsTwo round PCR was used for library preparation. Libraries were created from 450ng (equivalent of 70,000 DNA copies) of bone marrow DNA and 50ng of Human Genomic DNA (Roche). In the first round of PCR rearranged IgH genes were amplified using IGH FR3 BIOMED-2 primers (van Dongen, Leukemia 2003). In the second round the sequencing adapters and multiplex identifiers were added. Sequencing was performed on Ion Torrent PGM/Ion Proton sequencers using a 200bp chemistry. We developed a bioinformatics algorithm for detection of reads with known clonal V-D-J rearrangements within the resulting fastq files.For validation of the assay we sequenced 1E-1 to 1E-5 dilutions of diagnostic samples from 2 patients in multiplicates. The results show that the assay gives reproducible quantitative results up to 1E-4 dilution.ResultsWe sequenced 183 samples from 67 patients (52×day 15, 65×day 33, 66×day 78) with childhood ALL treated according to AIEOP-BFM ALL 2000 protocol with the median coverage 587,406 reads per sample. Eighty-three (45.4%) samples were negative by both methods. Fifteen (8.2%) samples were positive by NGS and negative by qPCR and 14 (7.7%) samples were positive by qPCR and negative by NGS. All the discordant samples had MRD levels below the sensitivity of both methods. The overall correlation of all double positive and double negative samples was very good (R2=0.93). If risk group stratification based on NGS results would be performed, 8 patients would be classified as intermediate risk (IR) instead of standard risk (SR) (one of whom relapsed) and 8 patients as SR instead of IR. One patient would be relocated from IR to slow early responders (SER) group, and two patients from SER to IR (one of them relapsed). One patient who relapsed would be classified as high risk (HR) instead of SER. All 5 patients who were MRD negative at d15 by NGS remained MRD negative in later timepoints and none of them relapsed.DiscussionWe present a cost-effective and widely adoptable NGS-based method that provides clinically relevant results in childhood ALL. NGS has a great potential to reduce the laboriousness associated with patient-specific qPCR analysis and to speed up the process of MRD detection. The sensitivity of both methods is comparable when ~500ng of DNA is used. The majority of the differences were in the samples with MRD levels below 1E-4 and most of treatment stratification changes occurred between SR/IR. However, the different stratification mostly concerned patients who did not relapse. The sensitivity of NGS could be improved if more DNA was analyzed. However, the benefit of increased MRD sensitivity is questionable due to possible overtreatment of patients with very low MRD loads after induction treatment. “Online” identification of d15 MRD negative patients reported as having an excellent prognosis previously is possible only by NGS, because optimization of patient-specific qPCR takes several weeks.The addition of 10% polyclonal DNA seems to solve the problem of MRD overestimation by NGS in samples with B-cell aplasia. At present, the main drawback of the Ig/TCR-exploring NGS methods is lack of standardization both in the experimental setting and in data analysis. Therefore, recently a European network, the EuroClonality NGS Consortium, has been formed to standardize the whole workflow of analytics, pre-analytics and bioinformatics not only for MRD quantification but also for clonality assessment in lymphoid neoplasms and for repertoire analysis.Supported by IGA NT14343, IGA NT12397, IGA NT13462-4 and GAUK 394214. DisclosuresNo relevant conflicts of interest to declare.

FIRST REPORT OF A "CANDIDATUS PHYTOPLASMA SOLANI" RELATED STRAIN ASSOCIATED WITH A POTATO REDDENING DISEASE IN GREECE

Potato (Solanum tuberosum), one of the most important crops worldwide, is susceptible to a high number of viruses and phytoplasmas. During the summer of 2013 symptoms similar to those caused by phytoplasmas, such as dwarfing, bright red discoloration, and rolling of the top leaves were observed in potato crops of cvs. Spunta and Jelly in Kastoria (40° 31'0 .12 "N21° 16'0 .12" E), Northern Greece. The affected plants were scattered in the field and their incidence ranged among the plots between 10% and 20%. In order to investigate the etiology of the aforementioned symptoms, leaves from 10 symptomatic and 10 asymptomatic plants of each variety were collected and subjected to DNA extraction. The extracts were tested using a generic nested PCR that targets the highly conserved 16S rRNA gene of phytoplasmas. More specifically, the universal primer set P1/P7 (Schneider et al., 1995) was used in the first round of PCR, whereas R16F2n/R16R2 primers (Gundersen and Lee, 1996) were applied in the nested step. The expected PCR products of ca. 1200 bp were amplified from all samples coming from symptomatic plants but not from the asymptomatic ones. One amplicon from each variety was further sequenced in both directions and their BLAST analysis revealed 98% similarity with the sequences of several “Candidatus Phytoplasma solani” (16SrXII-A stolbur group) strains (e.g. Bulgarian strain 241/13, KF907506). The two Greek sequences were 99% similar between them and were deposited in the EMBL-EBI under the accession Nos HG531809 and HG792592. “Ca. Phytoplasma solani” is endemic in Greece and was first detected in Datura stramonium plants in the area of Thessaloniki (Macedonia, Northern Greece). However, this is to our knowledge the first report of a potato disease caused by a “Ca. Phytoplasma solani” related strain in our country.