Purified Polymerase Chain Reaction Products Research Articles

Short-homology-mediated PCR-based method for gene introduction in the fission yeast Schizosaccharomyces pombe.

Schizosaccharomyces pombe is a commonly utilized model organism for studying various aspects of eukaryotic cell physiology. One reason for its widespread use as an experimental system is the ease of genetic manipulations, leveraging the natural homology-targeted repair mechanism to accurately modify the genome. We conducted a study to assess the feasibility and efficiency of directly introducing exogenous genes into the fission yeast S. pombe using Polymerase Chain Reaction (PCR) with short-homology flanking sequences. Specifically, we amplified the NatMX6 gene (which provides resistance to nourseothricin) using PCR with oligonucleotides that had short flanking regions of 20bp, 40bp, 60bp and 80bp to the target gene. By using this purified PCR product, we successfully introduced the NatMX6 gene at position 171 385 on chromosome III in S. pombe. We have made a simple modification to the transformation procedure, resulting in a significant increase in transformation efficiency by at least 5-fold. The success rate of gene integration at the target position varied between 20% and 50% depending on the length of the flanking regions. Additionally, we discovered that the addition of dimethyl sulfoxide and boiled carrier DNA increased the number of transformants by ~60- and 3-fold, respectively. Furthermore, we found that the removal of the pku70+ gene improved the transformation efficiency to ~5% and reduced the formation of small background colonies. Overall, our results demonstrate that with this modified method, even very short stretches of homologous regions (as short as 20bp) can be used to effectively target genes at a high frequency in S. pombe. This finding greatly facilitates the introduction of exogenous genes in this organism.

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.

Complete Genome Sequencing of an Embryonated Chicken Egg-Adapted Duck atadenovirus A.

Egg drop syndrome (EDS) is prevalent in industrial poultry globally. This disease is caused by Duck atadenovirus A or EDS virus (EDSV), a member of the genus Atadenovirus under the family Adenoviridae. The disease is attributed to significant economic losses in the poultry industry worldwide due to a drop in egg production, reduction in egg quality, and failure to reach maximum egg production. Oil-adjuvant inactivated vaccines, which are widely used in the poultry industry, provide good protection for immunized chickens against EDS. This study aimed to genetically and phylogenetically analyze the full-length genome of an embryonated chicken egg-adapted EDSV strain 127. After extraction of viral DNA from the allantoic fluid, overlapping fragments of the viral genome sequence were generated by polymerase chain reaction (PCR) using 25 pairs of primers. Purified PCR products were subjected to complete genome sequencing by the next-generation sequencing (NGS) approach. The nucleotide homology observed between genomes of the studied strain and that of the original strain 127 (NC_001813) of laying chickens was 99.9%. Its genome was 33,213 bp in length, with a G + C content of 43.01%. A comparison of the genome sequence of the egg-adapted virus with strain 127 revealed only three non-synonymous single-nucleotide polymorphisms (SNPs) between these viral genome sequences. Two mutations of S320G and I62K out of these SNPs were found within the coding regions of fiber and hypothetical proteins which may play a role in the adaptation of EDSV in the embryonated chicken eggs. The full genome sequencing of EDSV using NGS techniques provides insights into the discovery of genetic variants. Moreover, the genome sequence information of the EDSV provides valuable data for vaccine development in near future.

Impact of protein supplementation on semen quality, fertility, and BMP1R gene expression in sheep of Bangladesh.

A study was carried out to know the impact of protein supplementation on fertility and expressions of the fertility gene BMP1R. Three International Organization for Standardization (ISO), isocaloric but different levels of protein supplement ration (11.70% crude protein [CP] for control/To, 12.99% CP for T1, and 13.86% CP for T2) were fed to three different groups of sheep. DNA was extracted from the whole blood sample for polymerase chain reaction (PCR) of the BMP1R fertility gene, and purified PCR products were sequenced by a Sanger sequencer. Sequence alignment, pair, and multi-alignment comparison of the BMP1R gene of the species were done with MEGA6. The semen volume (1.0 mL), sperm counts (4.2 × 107 million), and percentage of normal (94.3%) and viable sperm (3.7%) were higher in treatment 2 than in the other two groups. The semen volume (1.0 mL), sperm counts (4.2 × 107 million), and the percentage of normal (94.3%) and viable sperm (3.7%) were higher in treatment 2 than in the other two groups. Ewes treated with supplemented, protein concentrate reached the conception at an earlier age (treatment 1, 9.5 ± 0.16 mo and treatment 2, 10.3 ± 0.04 mo) than control (9.8 ± 0.15 mo). The lambing interval varied, from 198 to 202 d. Lamb’s birth weights in three treated groups were ranging from 1.2 to 1.39 kg. The designated sequences of BMP1R gene revealed 100% homology with the sequence of Kazakh sheep. The present study indicated that the influence of nutrition on reproductive performance and genomic study will be helpful for the genetic improvement of low-productive sheep.

Genetic Diversity of Philippine Carabao (Bubalus bubalis) Using Mitochondrial DNA D-loop Variation: Implications to Conservation and Management

Developing rational conservation programs for Philippine carabao (PC) (Bubalus bubalis) requires knowledge on the pattern of genetic variability within and between populations of swamp buffalo. Studying the genetic diversity and phylogeographic structure is essential to understand the distribution of the Philippine swamp buffalo maternal haplotypes towards the PC conservation. This study aimed to determine the genetic diversity and the phylogeographic structure of PC using molecular approaches. D-loop of the mitochondrial DNA (mtDNA) on the PC B. bubalis (n = 107) were randomly selected from 23 populations across the islands of Luzon, Visayas, and Mindanao as major islands sub-groups. DNA was isolated from the whole blood, and the D-loop region of the swamp buffalo was amplified using the polymerase chain reaction (PCR). Purified PCR products were sequenced with the Applied Biosystems Automated 3730. Results showed that the phylogenetic analyses detected 16 mtDNA haplotypes observed with 12 variable sites and haplotype and nucleotide diversities of 0.695 ± 0.042 and 0.004 ± 0.001, respectively. This study also identified the delineation of swamp buffalo populations into major and minor groups. Among the major groups, 14 haplotypes were included, and sequences were incorporated in the maternal lineage A. Minor groups which fell in the maternal lineage B identified two new haplotypes. Thus, the current findings revealed the moderate mtDNA haplotype diversity and weak phylogeographic structure of the PC. In addition, the results of this study will serve as a vital starting point in planning effective strategies and prioritizing the genetic resources for PC conservation and management programs.

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.

Isolation, molecular identification, and phylogenetic evaluation of Cryptococcus neoformans isolated from pigeon lofts, Psittaciformes, and Passeriformes in Ahvaz, Iran

Cryptococcus neoformans, the main pathogen in immunocompromised patients, is a ubiquitous free-living fungus that can be isolated from avian excreta, soils, and plant material. This study was carried out to determine the infection rate of pigeon lofts, Passeriformes, and Psittaciformes in Ahvaz, the capital of Khuzestan province in Iran and to determine varieties of Cryptococcus neoformans (C. neoformans). The 80 samples were collected from pigeon lofts. Also, 163 feces of captive birds (Passeriformes and Psittaciformes) which kept in Ahvaz pet shops, and the 70 cloacal swabs of pet birds (Passeriformes and psittaciformes) referring to the department of avian medicine (the faculty of veterinary medicine of Shahid Chamran University of Ahvaz) were analyzed. The samples were directly inoculated on niger seed agar (NSA) and also enriched in brain heart infusion broth and then inoculated on NSA. Dark brown colonies suspected to C. neoformans subcultured on saborouds dextrose agar and pure cultures subjected to molecular (polymerase chain reaction (PCR)) diagnosis. For detection of C. neoformans, primer sets that targeting the CNLAC1 gene were selected and nested PCR was conducted. For identification of C. neoformans varieties, a primer set targeting the STR1 gene was selected. For more accurate confirmation, the purified PCR products of some isolates were also sequenced, and based on the gene sequences, all of the isolates belonged to C. neoformans variety grubii (var. grubii)(serotype A). Totally 16 out of 80 pigeon samples (20%) were contaminated with C. neoformans. The results in pigeons disclosed a 98.64% identity when compared with other strains of C. neoformans (CN1525, T4, and T1) which were previously deposited in GenBank from Italy and Thailand. Also, 21 out of 233 samples from Psittaciformes (9.01%) were contaminated with C. neoformans. The results in Psittaciformes disclosed a 99.7% identity when compared with other strains of C. neoformans (TIMM1313, IFM5882, CN1525, etc.) which were previously deposited in GenBank from Japan and Italy, etc. In the present study, the samples belonging to the passerine order were free of C. neoformans infection. According to the results, C. neoformans is prevalent in pigeon flocks and pet birds including Psittaciformes in the Ahvaz area, and should be considered by pigeon and captive bird breeders, veterinarians, and public health organizations in Ahvaz. The cryptococcus species isolated from captive birds and pigeons could be potential pathogens in humans.

Identification of Divergent Isolates of Banana Mild Mosaic Virus and Development of a New Diagnostic Primer to Improve Detection.

Banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus belonging to the Betaflexiviridae family. It infects Musa spp. with a very wide geographic distribution. The genome variability of plant viruses, including the members of the Betaflexiviridae family, makes their molecular detection by specific primers particularly challenging. During routine indexing of the Musa germplasm accessions, a discrepancy was observed between electron microscopy and immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) test results for one asymptomatic accession. Filamentous viral particles were observed while molecular tests failed to amplify any fragment. The accession underwent high-throughput sequencing and two complete genomes of BanMMV with 75.3% of identity were assembled. Based on these sequences and on the 54 coat protein sequences available from GenBank, a new forward primer, named BanMMV CP9, compatible with Poty1, an oligodT reverse primer already used in diagnostics, was designed. A retrospective analysis of 110 different germplasm accessions from diverse origins was conducted, comparing BanMMCP2 and BanMMV CP9 primers. Of these 110 accessions, 16 tested positive with both BanMMCP2 and BanMMV CP9, 3 were positive with only BanMMCP2 and 2 tested positive with only BanMMV CP9. Otherwise, 89 were negative with the two primers and free of flexuous virions. Sanger sequencing was performed from purified PCR products in order to confirm the amplification of the BanMMV sequence for the five accessions with contrasting results. It is highly recommended to use the two primers successively to improve the inclusiveness of the protocol.

First Report of Association of a ‘Candidatus Phytoplasma aurantifolia’ 16SrII-D Related Phytoplasma with Geranium (Pelargonium hortorum) Little Leaf Disease in Iran

Geranium, an important ornamental plant belong to the family Geraniaceae, is widely cultivated in urban landscapes and private gardens all over the world. During a survey in spring 2019, a few seed-propagated geranium plants exhibiting typical symptoms induced by phytoplasmas, including pale green and little leaves, phyllody, virescence, and witches’-broom symptoms, were observed in Jiroft, Kerman province of Iran. Total genomic DNA extracted from leaf midribs and petioles of the three symptomatic and two asymptomatic plants was used as a template in a nested polymerase chain reaction (PCR) assay using universal phytoplasma primer pairs P1/P7 followed by R16F2n/ R16R2 (Gundersen and Lee 1996) to amplify a part of the 16S rDNA phytoplasma genome as previously described by Azadvar et al. (2011). The generated 1.8- and 1.25-kb amplicons in the first and second round of the nested PCR assay, respectively, from each of the three symptomatic plant samples revealed phytoplasma infection of the plants. No amplicon was generated in PCR assays by using the DNA templates from symptomless plants. The purified PCR products were directly sequenced (Bioneer, South Korea) in both forward and reverse directions. The consensus sequences were analyzed by the iPhyClassifier tool (Zhao et al. 2009) and pDRAW32 software (AcaClone software, http://www.acaclone.com) for 16Sr group/subgroup phytoplasma identification. The 16S rDNA nucleotide sequences from the three diseased plants were identical (GenBank accession no. MT187639). iPhyClassifier analysis showed that the F2nR2 16S rRNA encoding gene sequence of the phytoplasma associated with geranium little leaf (GrLL) disease in Iran was identical (similarity coefficient 1.00) to those of the reference pattern of 16Sr group II subgroup D (GenBank accession no. Y10097) and had 98.7% similarity with the ‘Candidatus Phytoplasma aurantifolia’ reference strain (GenBank accession U15442). Virtual restriction fragment length polymorphism (in silico RFLP) analysis of the 16S rDNA F2nR2 sequence with the pDRAW32 program using AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, RsaI, Sau3AI, SspI, and TaqI endonucleases further confirmed that the GrLL phytoplasma belong to subgroup II-D. The 16SrII-D subgroup phytoplasma has been previously reported in association with Calendula officinalis (Esmailzadeh Hosseini et al. 2016), Helianthus annuus (Salehi et al. (2015), and Petunia hybrida (Faghihi et al. 2014) ornamental plants in Iran. We also found many petunia (P. hybrida) plants showing little leaf, yellowing, phyllody, and witches’-broom symptoms next to the GrLL-infected plants, which hosted the same phytoplasma (GenBank accession no. MT187637). A 16SrII group-related phytoplasma has previously been reported associated with Pelargonium capitatum weed plant exhibiting severe stunting and necrosis in Australia (Lee et al. 2010). To our knowledge, this is the first report of a ‘Ca. P. aurantifolia’ 16SrII-D subgroup-related phytoplasma associated with geranium (Pelargonium hortorum) little leaf disease in the world.

ASSESSMENT OF 16S rRNA SEQUENCE AND HETEROTROPHIC PLATE COUNT (HPC) METHODS OF IDENTIFYING BACTERIA FROM DRINKING WATER SYSTEMS IN BENIN CITY METROPOLIS

A variety of simple culture-based tests which are proposed to recover a wide range of microorganisms from water are collectively known as heterotrophic plate count (HPC) and used as an indirect indicator to give information about water quality. The aim of this study was to assess the heterotrophic plate count (HPC) culture-based dependent and 16S rRNA independent techniques of identifying bacteria. The HPC was conducted by incubating a filtered sample of water on R2A agar plates and enumerating the number of resultant bacterial colonies that grow on each plate. The molecular analysis was performed by extracting the deoxyribonucleic acid (DNA) from bacterial isolate and polymerase chain reaction (PCR) was done to obtain the amplicons (PCR products). Purified PCR products were sequenced by ABI V3.1 Big dye kit and the analysis of sequence was conducted by the basic local alignment search tool (BLAST) to identify their closest relatives. A total number of 17 isolates of Pseudomonas, Bacillus and Proteus were phenotypically identified, while the nucleotide sequence revealed the presence of 59 diverse strains with distribution (percentage) of Pseudomonas (25) 42.2 %, Bacillus 17 (28.8 %) and Proteus (17) 28.8 %. The investigated isolates when compared from gene data-base recorded genetic relatedness with similarity index of 61 to 100 %. The 16S rRNA gene sequences from DNA extractions showed microbial consortia in drinking water to comprise of a broad array of bacterial diversity, including those of critical concern to human health such as Pseudomonas and Bacillus. Finally, there was no significant correlation between the HPC test and 16S rRNA sequence analysis.

Rapid and sensitive detection of Shigella flexneri using fluorescent microspheres as label for immunochromatographic test strip.

Bacillary dysentery caused by Shigella genus is a major cause of morbidity and mortality worldwide. In China, the popular strain was mainly Shigella flexneri (S. flexneri). Therefore, fluorescent microspheres (FMs)-based immunochromatographic test strip (ICTS), as a novel, reliable, sensitive and uncomplicated method, was evaluated to detect S. flexneri. Sixty-three clinical samples of S. flexneri were collected in this paper. Polymerase chain reaction (PCR) combined with FMs-ICTS based on magnetic purification assay was developed for the quantitative detection of Shigella. And the genus-specific gene of ipaH and drug resistant gene of CTX-M-9 from Shigella were selected to investigate the potential of this new method. The sensitivity and specificity of this method were demonstrated by classical microbiological methods (API Coryne System), PCR assay based on agarose gel electrophoresis (PCR-GE) and the real-time fluorescent quantitative PCR (RTFQ-PCR) method. Under optimized conditions, the lower detection limits of PCR-ICTS, PCR-GE and RTFQ-PCR were 2.5×10-7, 2.5×10-5 and the 3.2×10-7 ng/µL, respectively. Experiments demonstrated the PCR-ICTS has a diagnostic agreement of 100% with conventional PCR and RTFQ-PCR on detection of clinical samples and could correctly recognize Shigella and non-Shigella from different microbial samples. After the purification of PCR products with Silicon coated magnetic nanoparticles (Si-MNPs), the false positive results were removed because of the strong screening ability of the purification process. Our results showed that FM-based ICTS was promising for measurable and sensitive detection of S. flexneri within 3 h. The results from immunochromatographic test were agreement with those from API Coryne system and RTFQ-PCR. Hence, this developed method might be useful for screening and monitoring clinical sample of S. flexneri, due to its speed, non-poisonous, simplicity and low-cost and helpful for promoting the prevention and control of communicable diseases caused by enteric pathogens such as S. flexneri.

First Evidence of Teladorsagia circumcincta Infection in Sheep from Egypt

Trichostrongylid nematodes are a common cause of gastroenteritis in sheep. Despite its worldwide distribution, Teladorsagia circumcincta has not been included in reports listing the various trichostrongyles infecting sheep from Egypt. Herein, we describe the presence of 2 T. circumcincta haplotypes infecting small ruminants from Egypt. For this study, fresh fecal samples were collected from 340 sheep and 115 goats reared at 5 districts in Dakahlia governorate and its surroundings, Egypt. Trichostrongyle eggs were harvested from the samples, and then subjected to DNA isolation and analysis. Polymerase chain reaction (PCR) amplification was carried out for the second internal transcribed spacer of ribosomal DNA (ITS2 rDNA). Purified PCR products of T. circumcincta were sequenced, and the revealed sequences were subjected to the nucleotide and phylogenetic analysis. A relatively high prevalence of trichostrongyles eggs was identified in sheep (33.2%) and a lower prevalence was found in goats (14.7%). Molecular analysis revealed, for the first time, 2 sheep herds from Egypt that were infected with T. circumcincta. Both infected herds were raised by the Bedouins in rural areas of El Mahalla El Kubra city. No T. circumcincta infections were found in any of the goats. Nucleotide sequence analysis revealed 2 haplotypes (Te1 and Te2) from 7 successfully sequenced samples (5 from the first and 2 from the second herd). Te1 was the major haplotype in both herds, and Te2 was retrieved from a single sample. Phylogenetic analysis displayed that the Te1 haplotype clustered with one from Cyprus, which might have been introduced to Egypt via goats imported from Cyprus due to a program to improve meat and milk production in Egypt. The present results could be beneficial in understanding the epidemiology of T. circumcincta and other trichostrongyles in Egypt, and have implementations in the effective control strategies used in this region.

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 novel apolipoprotein E mutation caused by a five amino acid deletion in a Chinese family with lipoprotein glomerulopathy: a case report

BackgroundLipoprotein glomerulopathy (LPG) is a rare kidney disease with a poor prognosis that is related to mutation of the apoE gene. More than 10 variants of apoE associated with LPG have currently been identified.Case presentationA male and his mother presented with proteinuria during a health examination. They went to hospital for further examination. Renal biopsy was performed, and the diagnosis was lipoprotein glomerulopathy (LPG), which is a rare, inherited renal disease. Medical histories were collected from the 2 LPG patients and their family members. The patients and family members underwent a routine urine test, and their renal function, blood lipids, and lipoprotein levels were examined. Genomic DNA was extracted from the peripheral blood of 7 family members, and exon 2, exon 3 and exon 4 of apoE were amplified by polymerase chain reaction (PCR). The purified PCR products were sequenced. Sequence analysis identified a 15 bp deletion (GCGCAAGCTGCGTAA) in exon 4 of the apoE gene that results in a novel 5 amino acid deletion in apoE (143 K-147R → 0). No mutations were found in exon 2 and exon 3 of the apoE gene.ConclusionsThis family study suggests that a novel ApoE mutation (143 K-147R → 0) may be etiologically related to LPG, and other genetic or environmental factors may be associated with the occurrence of LPG.

Isolation and molecular characterization of Mycoplasma spp. in sheep and goats in Egypt.

Background and Aim:Different species of Mycoplasma are associated with many pathological problems in small ruminants including respiratory manifestation, this problem results in significant losses, especially in African countries. This study aimed to (I) study some epidemiological aspects of Mycoplasma species infections in Egyptian sheep and goats at Giza Governorate, (II) diagnosis of Mycoplasma species affections using bacterial isolation and identification, (III) apply the polymerase chain reaction (PCR) for typing of different Mycoplasma species, and (IV) illustrate the phylogenetic tree for the isolated Mycoplasma species and other species from GenBank using the purified PCR product.Materials and Methods:A total of 335 samples were collected from sheep and goats from Giza Governorate in Egypt as 142 nasal swabs from clinically affected animals, 167 pneumonic lungs, 18 samples from tracheal bifurcation, and 8 samples by bronchial wash were cultured on pleuropneumonia-like organisms (PPLOs) media for cultivation of Mycoplasma species. PCR and sequencing and phylogenetic analysis were adopted to identify and classify the isolated Mycoplasma species.Results:A total of 24 Mycoplasma isolates were isolated on PPLO media, identified by biochemical tests, and confirmed and typed by PCR using specific primers. 10 isolates were confirmed as Mycoplasma arginini, four isolates as Mycoplasma ovipneumoniae by PCR, and 10 isolates as undifferentiated Mycoplasma species. A purified isolate of M. arginini and M. ovipneumoniae was sequenced and phylogenetic analysis was illustrated.Conclusion:M. arginini and M. ovipneumoniae are prevalent in Egyptian sheep and goats. Further studies on M. arginini are required due to its high frequency of isolation from pneumonic sheep and goats and also from animals suffer from different respiratory manifestations.

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 Blue Mold Caused by Penicillium oxalicum in Sweetpotato (Ipomoea batatas) in Korea

Many fungal diseases can affect sweetpotato (Ipomoea batatas [L.] Lam), a dicotyledonous plant in the Convolvulaceae family, at various stages from cultivation through storage (Clark et al. 2013). We observed blue mold disease on sweetpotato storage roots in the store house at the Bioenergy Crop Research Institute, Muan, South Korea, in 2016. The storage temperature and relative humidity were 13°C and 90%, respectively. Infected storage roots showed distinct rot symptoms, and bluish green sporulation was abundantly visible on the surface. Notably, disease was observed on two different local sweetpotato varieties, namely, Yulmi and Jinhongmi. Storage root samples showing rot symptoms were collected and used to isolate the causal organism. Fungi were isolated by the single spore isolation method onto potato dextrose agar (PDA). Repeated culturing of hyphal growth from the infected roots resulted in the isolation of two fungal isolates (SPL15027 and SPL15029). These were examined for morphological and molecular characteristics. Fungal growth on malt extract agar (MEA) at 25°C after 7 days appeared as a dull green center with pale green to white margins and lacked visible secreted pigmentation. The colony diameter on MEA was 37.9 ± 0.19 mm. Conidiophores were 57.3 to 205.2 (140) μm long, 2 to 4 metulae, 11.0 to 22.0 (17.4) μm, and phialides 7.2 to 20.0 (12.5) μm long in diameter. Conidia were globose to subglobose and were borne in columns. Conidial dimensions were 3.5 to 5.5 (4.2) × 2.5 to 4.0 (2.9) μm (n = 25). Morphologically, the pathogen was identified as Penicillium oxalicum Currie & Thom (Kozakiewicz 1992). Polymerase chain reaction (PCR) analysis was used for further molecular identification. Genomic DNA of the isolates was extracted using the Solg Genomic DNA Prep Kit (Solgent, Daejeon, South Korea), followed by PCR analysis using primers specific to the internal transcribed spacer (ITS5/ITS4) and β-tubulin locus (BT2a/BT2b) genes (Jing et al. 2015). Both strands of the purified PCR products were sequenced. These sequences were deposited in GenBank (ITS-KY853419/KY853420 and BT-KY853421/KY853422). BLAST analysis of the ITS (LT797553, KX515788, and HM469410) and BT (KF225064, AB849501, and KU600425) sequences showed 100% homology to P. oxalicum. This confirmed the identity of the isolated organism. Koch’s postulates were fulfilled by using the isolated culture for sweetpotato storage roots inoculations. PDA plugs (5 mm) and 50 μl of 2 × 10⁴/ml conidial suspension were used to infect different sweetpotato varieties including Chuhanmi, Yulmi, Jinhongmi, Yonhwangmi, and Dahomi. PDA plugs from blank plates and sterilized distilled water were used as respective noninoculated controls. Fungal-inoculated storage roots developed typical blue mold symptoms after 10 days of incubation at 25°C and under high humidity. In contrast, no symptoms were observed on noninoculated control storage roots. P. oxalicum was reisolated from the artificially inoculated roots, completing Koch’s postulates. To the best of our knowledge, this is the first report of P. oxalicum causing postharvest blue mold on sweetpotato in Korea.

Analysis of clinical phenotype and genotype of two hereditary coagulation factor XII deficiency

Objective Two Chinese pedigrees with congenital factor Ⅻ (FⅫ) deficiency were enrolled in the present study, and studies on the clinical manifestations, family survey, biochemical examinations and gene diagnosis of these pedigrees were performed. Methods In October 2014-2015 March, two cases of hereditary FⅫ deficiency patients were included in Xinhua hospital . Activated partial thromboplastin time(APTT), FⅫ procoagulant activity (FⅫ: C), FⅫ antigen(FⅫ: Ag)and other parameters of coagulant were detected. The FⅫ deficiency pedigree members, exons 1-14, boundary introns including the splice junctions of the F12 gene were amplified with polymerase chain reaction (PCR). Direct sequencing was exerted to purified PCR product to detect the gene mutation. If the gene mutations were found, polymorphism should be ruled out by directing sequence. One hundred and three healthy persons as normal controls. Results The two probands were manifested prolonged APTT (101 s and 143 s). They showed lower FⅫ activity and FⅫ antigen (2% and 6%, 0.4% and 4%, respectively). FⅡ: C, FⅦ: C, FⅧ: C, FⅨ: C, FⅩ: C and Fg are normal in the two probands. LAC is negative. Proband 1 has c. 1285C>T (p. Q429 stop) mutation. His parents and son have the heterozygous mutation in the same position. Proband 2 has c. 1556T>C(p.L519P)mutation. Her two sons have the heterozygous mutation in the same position. In the promoter regions of F12 gene, there were common 46C/T and 619 G/C polymorphisms in two pedigrees. Conclusion c. 1285C>T(p.Q429 stop)and c. 1556T>C(p.L519P)are the cause of FⅫ deficiency.(Chin J Lab Med, 2017, 40: 378-382) Key words: Factor Ⅻ deficiency; Factor Ⅻ; Mutation; Phenotype; Genotype; Pedigree

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.