Polymerase Chain Reaction Purification Research Articles

The Relationship between Gastroduodenal Pathologies and Helicobacter pylori cagL (Cytotoxin-Associated Gene L) Polymorphism.

The polymorphisms in the region between 58 and 62 amino acids of the 194-amino acid CagL protein (CagL hypervariable motif) affect the binding affinity of CagL to integrin α5β1 (ITGA5B1) receptor in host epithelial cells and have an effect on the development of various gastrointestinal diseases. We aimed to evaluate the associations of gastroduodenal pathologies, with the polymorphisms of cagL gene of Helicobacter pylori (H. pylori) and also associations between vacA genotypes and cagL polymorphisms. A total of 19 gastric cancer, 16 duodenal ulcer, and 26 non-ulcer dyspepsia patients were included in this case-control study. All cases had H. pylori. A fragment of 651 bp from gene cagL (hp0539) and cagA, vacA genes was amplified by polymerase chain reaction. Purified polymerase chain reaction products were sequenced by Sanger sequencing, and nucleotide sequences were translated into amino acid sequences. All of the H. pylori strains had cagL and cagA genes. In the 16 (84%) gastric cancer cases, the D58 amino acid polymorphism was significant than the 4 (15.4%) duodenal ulcer cases (P = .029), and the D58/K59 amino acid polymorphism was significant in 12 (63.1%) of the gastric cancer cases than 1 (3.85%) duodenal ulcer case (P = .008). D58/K59 and DKIGQ (n = 10; 52.63%) were the most common polymorphisms in the gastric cancer and were associated with the vacA genotype s1/m2, respectively (P = .022 and P = .008). The D58/K59 amino acid polymorphism was found to have a significant Odds Ratio (OR) value of 8.9 (P = .0017) in multivariate logistic regression analysis. The risk of gastric cancer development is 8.9 times higher with D58/K59 polymorphism.

Isolation of Cystic Echinococcosis Causative Agents of Echinococcus equinus and Echinococcus ortleppi Species from Humans in the Central Anatolia Region of Türkiye

Cystic Echinococcosis (CE) caused by Echinococcus granulosus sensu strico is neglected in Türkiye despite being one of the common diseases due to agriculture being at the forefront, low socioeconomic status and unhygienic animal slaughter. Considering the morbidity, mortality, and difficulties in treatment, more studies and precautions are needed regarding this disease. In this study, it was aimed to genotype Echinococcus isolated from CE patients in the Central Anatolia region. DNA isolation from tissue samples taken from 60 CE patients was performed using the QIAamp DNA FFPE tissue kit. Cytochrome c oxidase subunit gene region of Echinococcus was targeted and JB3/JB4.5 primers were used for genotyping. Polymerase chain reaction (PCR) products were purified according to the instructions for use of the QIAquick PCR purification kit. PCR products were prepared using the ABI Prism BigDye Terminator V3.1 Cycle sequencing kit and the nucleotide sequences in the samples were evaluated with the ABI 3100 sequencing device. The nucleotide sequences obtained in the study were analyzed using MetaPIGA2, MRBAYES v.3.1.2, phyogenetic analysis using parsimony, Unigen programs, maximum likelihood, Bayesian and parsimony methods. It has been found that 88.4% (53/60) of Echinococcus isolates were E.granulosus s.s. in this study. It has been genotyped as 41.7% (25/60) G1, 30.0% (18/60) G3 and 16.7% (10/60) G2 genotype. It has been determined that 6.6% (4/60) of the other Echinococcus isolates were E.equinus and 5.5% (3/60) were E.ortleppi. It was observed that E.equinus and E.ortleppi were isolated from atypically located cysts and from those living in rural areas. The E.equinus and E.ortleppi species were not found in CE patients living in urban areas. CE cases are common in the Central Anatolia region due to dog and cattle breeding, and the disease agent Echinococcus species vary. Genotyping of Echinococcus species is effective in the development of CE treatment and control strategies. Study results can play an active role in the fight against CE, which has formed the basis of the "one health" approach in the world and in Türkiye in recent years.

The impact of urine microbiota in patients with lower urinary tract symptoms

IntroductionInflammation and infection are causative factors of benign prostatic hyperplasia (BPH). Urine is not sterile, and urine microbiota identified by DNA sequencing can play an important role in the development of BPH and can influence the severity of lower urinary tract symptoms (LUTS).Materials and methodsWe collected mid-stream voided urine samples from BPH patients and control participants and stored them in a freezer at − 80 °C. All enrolled participants were requested to provide information about their clinical characteristics and complete the International Prostate Symptom Score (IPSS) questionnaire. Each step of the procedure, including the extraction of the genomic DNA from the urine samples; the amplification by polymerase chain reaction (PCR); PCR product quantification, mixing, and purification; DNA library preparation; and sequencing was performed with quality control (QC) measures. Alpha diversity was indicative of the species complexity within individual urine samples, and beta diversity analysis was used to evaluate the differences among the samples in terms of species complexity. Pearson’s correlation analysis was performed to calculate the relationship between the clinical characteristics of the participants and the microbiota species in the urine samples.ResultsWe enrolled 77 BPH patients and 30 control participants who reported no recent antibiotic usage. Old age, high IPSS and poor quality of life were observed in the participants of the BPH group. No significant differences were observed in the alpha diversity of the samples. In the beta diversity analysis, there was a significant difference between the microbiota in the samples of the BPH and control groups according to ANOSIM statistical analysis. On comparing the groups, the ten bacterial genera present in the samples of the BPH group in descending order of abundance were: Sphingomonas, Bacteroides, Lactobacillus, Streptococcus, Alcaligenes, Prevotella, Ruminococcaceae UCG-014, Escherichia_Shigella, Akkermansia, and Parabacteroides. Spearman’s correlation analysis revealed that urine samples showing the presence of the bacterial genera Haemophilus, Staphylococcus, Dolosigranulum, Listeria, Phascolarctobacterium, Enhydrobacter, Bacillus, [Ruminococcus]torques, Faecalibacterium, and Finegoldia correlated with a high IPSS, and severe storage and voiding symptoms (P < 0.05).ConclusionOur current study shows that dysbiosis of urine microbiota may be related to the development of BPH and the severity of LUTS. Further research targeting specific microbes to identify their role in the development of diseases is necessary and might provide novel diagnostic biomarkers and therapeutic options.

Multi-frequency impedance sensing for detection and sizing of DNA fragments

Electronic biosensors for DNA detection typically utilize immobilized oligonucleotide probes on a signal transducer, which outputs an electronic signal when target molecules bind to probes. However, limitation in probe selectivity and variable levels of non-target material in complex biological samples can lead to nonspecific binding and reduced sensitivity. Here we introduce the integration of 2.8 μm paramagnetic beads with DNA fragments. We apply a custom-made microfluidic chip to detect DNA molecules bound to beads by measuring Impedance Peak Response (IPR) at multiple frequencies. Technical and analytical performance was evaluated using beads containing purified Polymerase Chain Reaction (PCR) products of different lengths (157, 300, 613 bp) with DNA concentration ranging from 0.039 amol to 7.8 fmol. Multi-frequency IPR correlated positively with DNA amounts and was used to calculate a DNA quantification score. The minimum DNA amount of a 300 bp fragment coupled on beads that could be robustly detected was 0.0039 fmol (1.54 fg or 4750 copies/bead). Additionally, our approach allowed distinguishing beads with similar molar concentration DNA fragments of different lengths. Using this impedance sensor, purified PCR products could be analyzed within ten minutes to determine DNA fragment length and quantity based on comparison to a known DNA standard.

Molecular Screening of PAX2 Gene Polymorphism in Primary Vesicoureteral Reflux Patients in Taif Governorate, KSA.

&lt;b&gt;Background and Objective:&lt;/b&gt; Primary Nonsyndromic Vesicoureteral Reflux (PVUR) is a widespread genetic malformation and considered a prevalent Congenital Abnormality of the Kidney and Urinary Tract (CAKUT). Mutations in the &lt;i&gt;PAX2 &lt;/i&gt;gene have been associated with abnormalities in the kidney extending from CAKUT to oncogenic processes. The present study analyzes the &lt;i&gt;PAX2&lt;/i&gt; polymorphisms and their association with primary VUR in Saudi children patients from the Taif governorate. &lt;b&gt;Materials and Methods:&lt;/b&gt; Fifteen children with primary VUR were identified and screened for gene mutations in the &lt;i&gt;PAX2&lt;/i&gt; gene by direct sequencing method of purified Polymerase Chain Reaction (PCR) products of all exons to elucidate the correlation between &lt;i&gt;PAX2&lt;/i&gt; gene and VUR. &lt;b&gt;Results:&lt;/b&gt; Seven new variants have been defined. Three polymorphic missense variants in homozygous genotype form were found in intron 8 and detected in eight patients, One missense mutation was found in exon 10 in the site of transactivation domain and detected in ten patients and &lt;i&gt;in-silico&lt;/i&gt; analysis predicted it as a pathogenic one, Three mutations were found in exon 11 and detected in all patients as a compound homozygous. &lt;b&gt;Conclusion:&lt;/b&gt; &lt;i&gt;PAX2&lt;/i&gt;is important for normal kidney development and mutations in the gene possibly lead to disturbance in the protein structure and could be non-functional thus mutations in &lt;i&gt;PAX2&lt;/i&gt; may be one of the causes of PVUR in Saudi Arabia. Further investigation is necessary to understand the aetiology of disease and maybe other genes implicated in VUR.

First Report of Garlic virus B infecting Garlic in India.

Garlic (Allium sativum L.) is an economically important spice and vegetable crop grown throughout the world. Garlic viral disease complex caused by multiple virus infections is an important constraint in exploiting the potential yield of garlic. Among these viral pathogens, allexivirus (family Alphaflexiviridae) is the genus of viruses known for their degenerative effect on garlic yield. Their coexistence with other viruses, particularly potyviruses, has an adverse effect on garlic yield and quality (Perotto et al. 2010). During Sept 2018, while screening garlic germplasm accessions for the presence of allexiviruses, symptoms like foliar mosaic and curling were observed on accession G-204, planted at an experimental plot of ICAR-DOGR, Pune, India. A total of five samples comprised of five randomly selected G-204 garlic plants were collected from the experimental plot. Each sample contained leaves from the top, middle, and bottom portion of the individual garlic plants. These samples were subjected to RNA extraction using the RNeasy Plant Mini Kit (Qiagen, Germany) followed by reverse transcription (RT) using the Transcriptor cDNA synthesis kit (Roche Diagnostics, GmbH, Germany). The extracted RNA was then tested for allexiviruses such as garlic virus A (GarV-A), garlic virus B (GarV-B), garlic virus C (GarV-C), garlic virus D (GarV-D), and garlic virus X (GarV-X) by polymerase chain reaction (PCR) (Gawande et al. 2015; Roylawar et al. 2019; Baranwal et al. 2011; Gieck et al. 2009). Leaf samples tested through RT-PCR were found positive for garlic viruses GarV-A, GarV-B, GarV-C, GarV-D, and GarV-X. Allexiviruses other than GarV-B had been previously reported in India and hence further tests were conducted to confirm GarV-B infection. RT-PCR using primers, CF 5'- ATGGGAGACAGGTCGCAA-3' and CR5'- CTAAAATGTAAGCATGAGCGGT-3' designed specific to the coat protein yielded a 735-bp amplicon from all five G-204 plants. The amplified product was purified using QIAquick PCR Purification Kit (Qiagen, Germany) and cloned in pJET1.2 vector (Thermo Scientific, Lithuania). Two clones containing the CP gene were bidirectionally sequenced, and a consensus sequence was submitted to GenBank (MN650206). BLASTn results indicated that this consensus sequence showed 97.96% nucleotide (KP657919.1) and 100% amino acid sequence (AKN19940.1) identity with the CP sequence of GarV-B isolate from Poland. The presence of GarV-B was confirmed by enzyme-linked immunosorbent assay (ELISA) using a double-antibody sandwich ELISA kit (Arsh Biotech, Delhi, India) as per the manufacturer's protocol. An absorbance of reaction was read using a microplate reader at 405 nm. The mean OD values of negative and positive controls were 0.034 and 0.373, respectively. The OD values of five samples tested ranged from 0.210 to 0.296 indicating a positive reaction for GarV-B. To assess the presence of GarV-B in the available genetic stock, we tested 30 garlic germplasm accessions for GarV-B using RT-PCR. Out of these, 17 accessions were found positive for GarV-B. GarV-B has been reported from many countries (Gieck et al. 2009). This is the first report of GarV-B from India. Globally, allexiviruses are known for their adverse impact on garlic production (Oliveira et al. 2014). GarV-B together with other viruses can be a potential threat to garlic production in India. Further, detailed evaluations are needed to study the impact of GarV-B on garlic production in India.

Sequence analysis of the Hex A gene in Jacob sheep from Bulgaria.

Background and Aim:Jacob sheep are a rare ancient breed of sheep believed to have originated from the Mediterranean area but which are now kept throughout the world. These sheep have recently attracted medical interest due to the observation of a genetic disorder in the breed that can be used as an animal model of Tay–Sachs disease (TSD). This study aims to detect mutations in the Hexosaminidase A gene in Jacob sheep based on sequence analysis of the 284-bp fragment situated between exon 11 and intron 11 of the gene, a target sequence for site-specific mutation. This is the first study that has investigated Jacob sheep in Bulgaria for gene-specific mutations.Materials and Methods:A total of 20 blood samples were collected from Jacob sheep from the Rhodope Mountains. DNA was isolated from these samples, and a specific 284-bp fragment was amplified. The amplified products were purified using a polymerase chain reaction purification kit and sequenced in both directions.Results:Target sequences were successfully amplified from all 20 investigated sheep. Sequence analysis did not show the homozygous, recessive, missense (G-to-C transition) mutation at nucleotide position 1330 (G1330→C) in exon 11, demonstrating that all of these sheep were a normal genotype (wild-type).Conclusion:Jacob sheep are considered a potentially useful animal model in advancing the understanding of pathogenesis and developing potential therapies for orphan diseases, such as those characterized by mutant GM2 gangliosides. The clinical and biochemical features of the Jacob sheep model of TSD represent well the human classical late-infantile form of this disorder, indicating that the model can serve as a possible new research tool for further study of the pathogenesis and treatment of TSD.

First Report of Strawberry (Fragaria × ananassa) as a Host of a ‘Candidatus Phytoplasma americanum’-Related Strain in the United States

In June 2018, while conducting a survey for possible presence of phytoplasma pathogens in Pennsylvania production fields, a strawberry plant (Fragaria × ananassa) was observed with symptoms of stunting and unseasonal reddening and distortion of leaves. Only one out of 500 strawberry plants expressed the described symptoms in a farm in Perry County, PA. Several leaves and stems were collected from the symptomatic plant as well as from a few asymptomatic ones for further work. Total DNA was extracted from 100 mg of leaf midveins with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA). All DNA samples were initially tested using a phytoplasma universal real-time polymerase chain reaction (PCR) assay (Hodgetts et al. 2009). Positive amplification results were obtained only with samples derived from the symptomatic strawberry plant. To detect and identify the suspected phytoplasmal pathogen strain(s), the DNA samples were used as a template in conventional PCRs for amplification of phytoplasma 16S rRNA, secY, secA, and tuf gene sequences as previously described (Dickinson and Hodgetts 2013; Lee et al. 2004, 2010; Makarova et al. 2012). All of the obtained amplicons were of expected sizes, and no amplification was observed from the asymptomatic strawberry samples. PCR products were purified with a QIAquick PCR Purification Kit (Qiagen) and sequenced. Nucleotide sequences of the PCR products were deposited in the GenBank database under accession numbers MN227133 to MN227136. The nucleotide sequences were analyzed using Geneious software (Biomatters, Auckland, NZ) for sequence alignments and using iPhyClassifier (Zhao et al. 2009) for group-subgroup and ‘Candidatus Phytoplasma’ species assignments. The classification into 16Sr groups and subgroups was established by virtual restriction fragment length polymorphism (RFLP) analysis of PCR products compared with reference strains. The phytoplasma, termed strain SRL1-PA (Strawberry Red Leaf1-PA), was found to share 99.9% similarity with that of the ‘Candidatus Phytoplasma americanum’ reference strain (GenBank accession DQ174122). The virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment is most similar to the reference group XVIII, subgroup A (GenBank accession DQ174122), with a pattern similarity coefficient of 0.98. Another sample from the same strawberry plant was independently determined positive for ‘Ca. P. americanum’ by CPHST Beltsville Lab using nucleotide sequence analysis of 16S rRNA gene sequences amplified in end point PCR. Alignments and phylogenetic analyses of secY, secA, and tuf gene sequences confirmed the close relatedness of the Pennsylvania SRL1-PA strain with the reference strains of ‘Ca. P. americanum’. ‘Ca. P. americanum’ was previously reported (Lee et al. 2006) in Texas and Nebraska on potato and associated with potato purple top wilt disease. In 2019, the Pennsylvania Department of Agriculture team revisited the same farm and collected additional samples including some from potato but was not able to detect any phytoplasma. To our knowledge, this report provides the first evidence for the presence of a ‘Ca. P. americanum’-related strain in Pennsylvania and more importantly describes strawberry (Fragaria × ananassa) as a new host for this phytoplasma. With only one single positive strawberry plant in a single year, the true extent of disease spread on strawberries remains undetermined.

First Report of Apple Hammerhead Viroid Infecting Apple Trees in South Korea

First Report of Apple Hammerhead Viroid Infecting Apple Trees in South Korea

First Report of Anthracnose of Persimmon Caused by Colletotrichum nymphaeae in Korea

Jiro Asian persimmon (Diospyros kaki L. f.) is widely found growing in houses, in gardens, and on roadsides in South Korea. Many factors including fungal diseases such as anthracnose negatively impact the yield and quality of persimmon crops. In July 2018, serious cases of anthracnose disease were observed in gardens in Sanju, South Korea. The disease incidence was >40% (based on five trees observed). Green persimmon fruits were affected by anthracnose the most. Lesions on infected fruits were sunken, brown, and round (∼6.1 mm in diameter). Presumed causative agents were isolated from the lesions of infected fruits. Small pieces of tissue were cut from the lesions with a sterile scalpel, disinfected with 1% sodium hypochlorite solution for 1 min, rinsed twice with sterile distilled water, and dried by blotting. Pieces of the tissue were placed on water agar and incubated at 25°C in the dark. Newly emerging fungal colonies from the tissue samples were transferred onto potato dextrose agar and incubated at 25°C in the dark. Fungal colonies were purified using a single-spore isolation technique (Cai et al. 2009). Two fungal colonies were used for examining morphological characteristics. Seven-day-old colonies were gray on their upper side and creamy white on their bottom side. Conidia hyaline and fusiform with pointed ends, measuring 10.5 to 18.0 × 4.6 to 7.8 (n = 50; mean ± SD = 14.4 ± 1.5 × 6.4 ± 0.6). Conidia were produced across the entire colony. Appressoria oval, cylindrical, lobed, medium brown, seen singly or in chains, measuring 9.0 to 12.9 × 6.0 to 9.0 µm (n = 25; mean ± SD = 10.9 ± 1.5 × 7.2 ± 1.0 µm). The morphological characteristics of the present isolate are consistent with several Colletotrichum species within the Colletotrichum acutatum species complex, including Colletotrichum nymphaeae (Damm et al. 2012). The representative isolate PNKU18D1 was used in molecular and phylogenetic analysis. Five target gene regions including the ITS, TUB2, GAPDH, CHS-1, and ACT regions were amplified from genomic DNA. The primer sets ITS1/ITS4, Bt2a/Bt-2b, GDF/GDR, CHS-79F/CHS-354R, and ACT512F/ACT783R were used to amplify ITS, TUB2, GAPDH, CHS-1, and ACT, respectively (Damm et al. 2012; Weir et al. 2012). Purified polymerase chain reaction products were sequenced by Macrogen (Seoul, Korea) and deposited in GenBank (accession nos. LC428839 to LC428843). Neighbor-joining and maximum likelihood analyses were performed using MEGA6 based on the multilocus alignment (ITS, GAPDH, CHS-1, ACT, and TUB2). Analyses showed that the isolate clustered with the C. nymphaeae strain with high bootstrap support (>98%). The BLAST results using each gene sequence also revealed a 99 to 100% match with respective gene sequences in C. nymphaeae GenBank databases. The virulence of the PNKU18D1 isolate was tested in green persimmon fruit. The surfaces of healthy green fruits were sterilized with 70% ethanol and washed twice with distilled water. Eight fruits were inoculated with 10 µl of conidia suspension (10⁵ conidia/ml) placed on a single point on each fruit. Eight fruits inoculated with distilled water were used as controls. Inoculated fruits were placed in a large plastic box on a sterile plastic saucer and incubated at 25°C in the dark at high humidity for 1 week. The experiments were repeated twice. Typical anthracnose symptoms including necrotic lesions (5.5 ± 0.6 mm in diameter) were observed in 90% of inoculated fruits. Control fruits were asymptomatic. The fungus was then successfully reisolated and identified as C. nymphaeae according to the methods described above, thus satisfying Koch’s postulates. C. gloeosporioides, C. acutatum, C. siamense, and C. horii have been previously reported as the causal agents of persimmon anthracnose in Korea (Hassan et al. 2018; Kwon et al. 2013), and in this study we provide evidence of anthracnose resulting from infection by C. nymphaeae. To the best of our knowledge, this is the first report of C. nymphaeae causing anthracnose in persimmon in South Korea.

A two‐buffer method used for both polymerase chain reaction product purification and DNA gel extraction

AbstractBACKGROUNDBoth polymerase chain reaction (PCR) purification kits and DNA gel extraction kits have been widely used for DNA purification in laboratories worldwide. However, the uses of these kits are costly and produce laboratory wastes that have negative impacts on the environment; it is important to develop a novel method to overcome these drawbacks.RESULTSHere we demonstrate that a saturated sodium iodide solution and a laboratory‐made washing buffer can replace the buffers in PCR purification and gel extraction kits for DNA purification and do not affect DNA purification capacity. These two buffers can be utilized to purify DNA along with the columns recycled from PCR purification and DNA gel extraction kits and can maintain DNA purification capacity that is comparable to that of commercial kits. For small‐sized DNA fragments, the buffers developed in this study are even more efficient compared to commercially available buffers from PCR purification and DNA gel extraction kits. The quality of DNA purified with these two buffers can meet the requirements of gene cloning.CONCLUSIONSThis study provides a two‐buffer method that is used for both PCR product purification and DNA gel extraction. This method is simple, of low cost and beneficial to numerous laboratories worldwide. © 2019 Society of Chemical Industry

Detection of ERG11 point mutations in Iranian fluconazole-resistant Candida albicans isolates.

Background and Purpose:Candidiasis is referred to a group of superficial and deep-tissue fungal infections often caused by Candida albicans. The superficial infections affect the oral, oropharynx, esophagus, and vaginal mucosa. The treatment of choice for these infections is the use of azoles, such as fluconazole. However, the increased use of these antifungal agents has led to the emergence of azole-resistant isolates of C. albicans. Different mechanisms have been suggested for the development of drug resistance, such as mutations in the encoding gene ERG11. Mutations in ERG11 result in changes in the ERG11p spatial construction and reduce the affinity between the protein and azole. This study aimed to determine the susceptibility profile of C. albicans clinical isolates to fluconazole using microdilution method. The present research was also targeted toward the detection of mutations that might be related to fluconazole resistance by the amplification and sequencing of ERG11 gene.Materials and Methods:This study was conducted on a total of 216 clinical isolates obtained from Mashhad, Isfahan, and Tehran cities in Iran, during 2016-2018. The clinical isolates were identified using molecular techniques. Furthermore, minimum inhibitory concentration (MICs) was determined according to the clinical and laboratory standards institute M27-A3 and M27-S4 documents. The concentration range for fluconazole was obtained as 0.063-64 μg/ml. In the resistant strains, ERG11 genes were amplified by specific primers. Subsequently, cycle sequencing reactions were performed on purified polymerase chain reaction (PCR) products in forward and reverse directions. Finally, the results were analyzed by MEGA (version 7) and Gene Runner software (version 6.5.30).Results:Out of 216 strains, 100 (46.3%) species were identified as C. albicans. The MIC values for fluconazole had a range of 0.125-16 μg/ml with the MIC50 and MIC90 values of 0.5 and 1 μg/ml, respectively. Totally, 41 nucleotide changes were detected among 4 resistant isolates. In this regard, 4 out of 41 mutations in codons caused changes in ERG11p; however, these mutations did not lead to fluconazole resistance.Conclusion:Fluconazole resistance among clinical isolates is not merely due to the changes in ERG11p. This resistance may be also related to some other mechanisms, such as the prevention of the intracellular accumulation of the antifungal agent and alteration of the target enzyme to diminish drug binding.

First Report of Anthracnose Crown Rot of Strawberry Caused by Colletotrichum siamense in Rajshahi District of Bangladesh

Strawberry (Fragaria × ananassa) was introduced to Bangladesh in 1996 and has gained remarkable acceptance. However, with the expansion of acreage, anthracnose crown rot (ACR) is posing a challenge to strawberry growers. In March 2016, typical ACR symptoms were observed on cultivar Strawberry Festival in a farmer’s field in Rajshahi District. Symptoms were evident on approximately 10% of plants and included flagging of young leaves, mimicking lack of water, and wilting of the entire plant in daytime heat. A lengthwise cut through the crown of diseased plants showed characteristic shades of red and white marbling similar to that described by Weir et al. (2012). Crowns of seven symptomatic strawberry plants were obtained after excising foliage and roots and washed under running water for 1 h. Crowns were then surface disinfested with 70% ethanol followed by 1% NaClO for 2 and 3 min, respectively. Small pieces (2 × 2 mm) of the internal infected marbled red tissues were cut with a sterile scalpel, placed on corn meal agar, and incubated for 7 days at 25°C. Conidia grown on tissue pieces and hyphal mats from the tissues were streaked on PDA plates to obtain single conidial colonies. Two of the single-conidial isolates (BRSP08 and BRSP09) that had white margins with circular, dull orange centers were selected to prove Koch’s postulates. The average mycelial growth rate was 9.8 mm/day at 25°C on PDA with cottony aerial growth and pale yellowish to pinkish color. Conidia were hyaline, cylindrical, with round ends, and measuring 14.90 to 18.19 × 4.05 to 6.20 μm (average ± SD, 16.22 ± 1.20 × 5.90 ± 0.84 μm) (n = 30). Appressoria were brown, globose to cylindrical, lobbed, and measuring 7.50 to 12.70 × 6.08 to 8.4 μm (10.72 ± 1.30 × 7.20 ± 0.80 μm) (n = 20). The morphological characteristics of these isolates are consistent with some Colletotrichum species within the C. gloeosporioides complex including C. siamense (Afanador-Kafuri et al. 2003; Weir et al. 2012). BRSP09 was used for molecular identification. Internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) and genes encoding chitin synthase (CHS-1), actin (ACT), β-tubulin (TUB2), calmodulin (CAL), and the Apn2–Mat1–2 intergenic spacer and partial mating type (Mat1–2) gene (ApMat) of the selected isolate were amplified using primers ITS1F/ITS4, Bt2a/Bt-2b, CHS-79F/CHS-354R, ACT512F/ACT783R, CL1C/CL2C, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). Sequencing of the purified polymerase chain reaction products was performed by Macrogen (Korea), and sequences were deposited in GenBank (LC420017 to LC420022). Neighbor-joining and maximum likelihood phylogenetic trees were inferred using MEGA 6.0 (Tamura et al. 2013) based on the multilocus alignment (ITS, TUB2, ACT, CHS-1, CAL, and ApMat) of the present isolate and related Colletotrichum spp. sequences previously deposited in GenBank. BLAST search and phylogenetic analysis revealed that BRSP09 is C. siamense (Mills et al. 1992). To confirm the pathogenicity of the fungus, ten 3-month-old tissue-cultured Strawberry Festival plants were inoculated by dipping the roots and crowns into either 300 ml of conidial suspension (8 × 10⁶ conidia/ml) of the isolates BRSP08 and BRSP09 or sterile distilled water (control) for 30 min. The inoculated plants were transplanted into plastic pots with artificial peat-based soil mix and maintained in the greenhouse with 28/20°C day/night temperature. The experiment was conducted twice. After 4 weeks, all plants inoculated with BRSP08 or BRSP09 were stunted and developed typical wilt symptoms similar to those observed in the field, whereas the control plants remained healthy. C. siamense was reisolated from crown tissues with typical ACR symptoms and identified using morphological and molecular methods mentioned above, fulfilling Koch’s postulates. This is the first report of ACR of strawberry caused by C. siamense and the members of C. siamense complex in Rajshahi District.

First Report of Grapevine Red Blotch Virus in Mexico

Grapevine red blotch virus (GRBV) (Al Rwahnih et al. 2013) is a member of the recently recognized genus Grablovirus in the family Geminiviridae (Varsani et al. 2017). GRBV is the causal agent of grapevine red blotch disease (Yepes et al. 2018), affecting viticulture in North America and some other regions around the world (reviewed in Sudarshana et al. [2015]). In this study, we sampled grapevine plants displaying red blotch symptoms in the major wine producing region of Mexico: Ensenada, Baja California, in 2016 and 2017. A total of 46 samples from cultivars Vitis vinifera L. ‘Pinot Noir’ and ‘Merlot’ were tested in 2016 by end-point polymerase chain reaction (PCR) using primers GRBV-V2F (5′-ATGGGTTAGGGGATGAGGCT-3′) and GRBV-V1R (5′-CGGCAATGACTCCTGCGGCT-3′). Results showed amplification of the expected 798-bp product from 31 samples. One of these amplicons was cloned, sequenced, and compared with reported sequences through a BLASTn search. The fragment had high nucleotide sequence identity (98%) with GRBV isolate BCRB5 from Canada (GenBank accession no. KX234092). The presence of GRBV was corroborated using the AmplifyRP Acceler8 kit (Agdia, Elkhart, IN), which detects the GRBV genome by isothermal recombinase polymerase amplification (RPA). In 2017 symptomatic V. vinifera ‘Nebbiolo’ plants were tested by multiplex PCR to amplify a region of the replicase-associated protein (Rep) gene with primers Repfor/Reprev (expected product 318 bp) and a fragment from the coat protein (CP) gene with primers CPfor/CPrev (expected product 257 bp) (Krenz et al. 2014). Amplicons from a positive sample were cleaned using a GenJet PCR purification kit (Thermo Fisher Scientific, Waltham, MA) and analyzed by Sanger sequencing. Both Rep and CP fragments shared >99% nucleotide sequence identity with GRBV isolates ONRB7 (KY316025) and ONRB4 (KY316022) from Canada (Poojari et al. 2017). To gain a deeper insight into the phylogeny of local GRBV isolates, we sequenced the full-length viral genome of 2016 and 2017 representative isolates. Each genome was amplified by high-fidelity PCR in two fragments, combining primers CPfor/Reprev (expected product 1.8 kb) and Repfor/CPrev (expected product 1.9 kb). After Sanger sequencing, the genomes were assembled in the GeneStudio sequence analysis software version 2.2.0.0 (GeneStudio, Suwanee, GA). A phylogenetic tree constructed after comparing the local isolates GRBV-Gpe-JGB (MH557096) and GRBV-Gpe-JCT (MH557095) to GRBV isolates from Canada and the United States grouped the Baja Californian isolates with clade 1 isolates. To our best knowledge, this is the first report of GRBV in Mexico.

Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays.

BackgroundPolymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process.Methodology/Principal findingsWe assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately.Conclusions/SignificanceThis is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.

Teaching Molecular Microbiology: Next Generation Sequencing vs Multiplex PCR

<h3>ABSTRACT</h3> Experience in Next Generation Sequencing (NGS) is a highly desirable skill for molecular technologists. Previously, our Molecular Pathology program partnered with a local laboratory affiliate to develop a hands-on NGS educational protocol. Recently, we expanded this instructional laboratory to include the BioFire FilmArray instrument, a sample-to-answer multiplex polymerase chain reaction (PCR) platform. We used these two methods to illustrate the strengths and weaknesses of each technology and to reinforce the principles of molecular microbiology. After several classroom lectures, 18 students were given a protocol to prepare an NGS DNA library from unknown bacterial isolates. Each student performed DNA isolation, PCR of 16S rRNA, and magnetic bead PCR purification. The samples were given to the affiliate laboratory for loading and analysis on the Life Technologies Ion Torrent NGS platform. Meanwhile, the students were paired up to perform the FilmArray gastrointestinal assay on their pooled bacterial isolates. Students compared the quantitative results of NGS with the qualitative results of the FilmArray, and they were able to determine which bacteria were detected by each method as compared with the key. There was high correlation between the two methods, although some false negatives and false positives were observed for each assay. These observations were used for classroom discussions about differences in microbial DNA isolation efficiencies, test menus, analytical sensitivities, and procedural limitations of each method. In sum, the integration of the NGS and multiplex PCR methods into a unified laboratory unit provided valuable hands-on technical experience while simultaneously illustrating the concepts of molecular microbiology.

Heterogeneous DNA methylation status in same-cell subpopulations of ovarian cancer tissues.

This study aims to explore the heterogeneous DNA methylation differences between individual single ovarian cancer cells isolated from the same formalin-fixed and paraffin-embedded human ovarian cancer tissue. Single cells were isolated by laser microdissection. Whole genome amplification and polymerase chain reaction purification were performed on the converted genomic DNA. Target primers designed for checking DNA methylation were used in polymerase chain reaction reactions to amplify special fragments. Sequencing was performed to analyze the heterogeneous DNA methylation statuses of different single ovarian cancer cells. Three of nine single human ovarian cancer cells showed positive bands (33.3%) on separating gel. The methylated and unmethylated CpGs were shown at the same loci in different single cells. We show heterogeneous DNA methylation statuses in same-cell subpopulations.

Association of Three Single Nucleotide Polymorphisms in MTR and MTRR Genes with Lung Cancer in a Turkish Population.

Folate metabolism plays a critical role in DNA methylation and synthesis. Polymorphisms in folate metabolism may affect enzyme activities and thereby affect the cancer risk. Methionine synthase (MTR) and methionine synthase reductase (MTRR) are critical enzymes for the folate cycle. In this study, possible associations between genetic variabilities in MTR and MTRR and susceptibility to lung cancer (LC) were investigated in a Turkish population. A case-control study with 193 LC cases and 199 noncancerous controls was conducted. DNA was extracted from leukocytes using the high pure polymerase chain reaction (PCR) template preparation kit. The MTR 2756 A>G (rs1805087), MTRR 524 C > T (rs1532268), and MTRR 66 A>G (rs1801394) genotypes were determined using PCR-restriction fragment length polymorphism (PCR-RFLP) assays. The genotype and haplotype analyses of these polymorphisms were performed using SPSS 21 and Haploview 4.2, respectively. An association between the MTRR A66G polymorphism and LC (p = 0.042) was found. In addition, this allele was observed more frequently in smokers compared to nonsmokers (p = 0.030). In contrast, the distribution of the MTR 2756 A>G and the MTRR 524 C > T allele frequencies were similar in the subject cases and controls. In conclusion, the present study suggests an association between the MTRR 66 A>G gene polymorphisms and LC risk in a Turkish population.

Associations between single-nucleotide polymorphisms of melanocortin gene and sexual desire behavior in rabbit (Oryctolagus cuniculus)

Understanding the genetic basis of polymorphisms which can affect the sexual desire behavior of rabbit doe is important. This study was designed to investigate the association of 2 parts of melanocortin gene (MC4R-1, MC4R-2) polymorphism and the sexual desire behavior for V-line rabbits. V-line does were divided according to their ability to mate into group 1 “receptive does” (high sexual desire) and group 2 “nonreceptive does” (low sexual desire) to identify DNA markers useful for association studies with the sexual desire behavior. DNA from blood samples of each group was extracted to amplify the MC4R gene. The purified polymerase chain reaction products were sequenced in those who had the highest and lowest sexual desire in rabbit line. Alignment of sequence data from each group revealed the following results: there is a variation detected in MC4R-1 between the 2 groups at nucleotide 35 (T-G) (sense mutation) while we detected another variation at nucleotide 19 (T-C) in MC4R-2 gene (sense mutation) of low sexual desire does. The results of average individual body weight of does for the 2 groups indicated that does in group 1 had significantly (P ≤ 0.05) lower body weight than does in group 2. The analysis of individual body weight and sexual desire behavior of does revealed a significant association between MC4R polymorphism and the sexual desire behavior in rabbits.

Associations between single nucleotide polymorphisms in multiple candidate genes and body weight in rabbits

Aim:In this study, we examined parts of six growth genes (growth hormone [GH], melanocortin 4 receptor [MC4R], growth hormone receptor [GHR], phosphorglycerate mutase [PGAM], myostatin [MSTN], and fibroblast growth factor [FGF]) as specific primers for two rabbit lines (V-line, Alexandria) using nucleotide sequence analysis, to investigate association between detecting single nucleotide polymorphism (SNP) of these genes and body weight (BW) at market.Materials and Methods:Each line kits were grouped into high and low weight rabbits to identify DNA markers useful for association studies with high BW. DNA from blood samples of each group was extracted to amplify the six growth genes. SNP technique was used to study the associate polymorphism in the six growth genes and marketing BW (at 63 days) in the two rabbit lines. The purified polymerase chain reaction products were sequenced in those had the highest and lowest BW in each line.Results:Alignment of sequence data from each group revealed the following SNPs: At nucleotide 23 (A-C) and nucleotide 35 (T-G) in MC4R gene (sense mutation) of Alexandria and V-line high BW. Furthermore, we detected the following SNPs variation between the two lines: A SNP (T-C) at nucleotide 27 was identified by MC4R gene (sense mutation) and another one (A-C) at nucleotide 14 was identified by GHR gene (nonsense mutation) of Alexandria line. The results of individual BW at market (63 days) indicated that Alexandria rabbits had significantly higher BW compared with V-line rabbits. MC4R polymorphism showed significant association with high BW in rabbits.Conclusion:The results of polymorphism demonstrate the possibility to detect an association between BW in rabbits and the efficiency of the used primers to predict through the genetic specificity using the SNP of MC4R.