Specific Primers Research Articles

Genotyping of Hydatid Cyst Isolated from Human and Domestic Animal in Babylon Province

The study involved 200 feces samples of cattle and sheep, in Babylon Governorate in Iraq. Used for diagnosis of hydatid cyst, polymerase chain reaction (PCR), using specific primers and the method was the conventional PCR. Cattle and sheep was classified into subgroups according to age, area and sex in this study association of these risk factors with rate of infection. The diagnosis of hydatid cyst in cattle and sheep was determined by (pcr). study which are collected from human, sheep, cattle The prevalence of Hydatid Cysts infections was highest in Al-Qassim and western hamza districts which were 36.36b % (40) of cattle, 22.5a% (9) of sheep and 17.5a % (7) of cattle , 22.72b% (25) of sheep respectively , while the prevalence of Hydatid Cysts in Al-Hilla District was 18.18ab% (20) of cattle, 37.5c% (15) of sheep and the lower prevalence of was in Al-Musayyab and Al-mahaweel district which was 9.09a % (10) of cattle, 12.5ab % (5) of sheep and 13.63ab % (15) of cattle , 10b % (4) of sheep respectively. The results of present study were exhibited on high prevalence of in cattle and sheep of some districts in Babylon province. The statistical analysis recorded that there was Significant difference (at P<0.05) difference in the prevalence of parasite among geographical areas. In the present study the incidence of hydatid cysts was different according to age of the host. In sheep the incidence of hydatid cysts has a positive proportion to age of sheep; a high prevalence rate (45b%) was observed in age of sheep (1-2 years); while less prevalence rate observed in (>3) years ages (10a%),In cattle a higher rate (58.18b%) was recorded at ages (2-5 years) while a lower rate (3.63a%) was recorded in (>8) years with Highly significant difference at P<0.05 difference in the prevalence of parasite between age intervals. The result of infection with hydatidosis according to sex revealed that Females were exposed to infection more than males, with no significant difference at P<0.05 in sheep and cattle.

Molecular Characterization of Human Adenoviruses Associated with Pediatric Respiratory Infections in Omdurman, Sudan

Acute respiratory viral infection (ARVI) is a common cause of morbidity and mortality especially in pediatric patients. Among important respiratory pathogens are adenoviruses, The illnesses can range from the common cold to pneumonia Depending on the type, adenoviruses can cause other illnesses such as gastroenteritis, conjunctivitis, cystitis, and, less commonly, neurological disease. Aim of study: molecular detection of respiratory infection due to adenovirus among children in Khartoum state. Respiratory specimens were collected from children’s patients with RTIs in the emergency department Mohamed Alamin Hamid pediatric hospital Omdurman in Khartoum state during the period of October 2022 to February 2023.the HAdV infection were determined by PCR technique. A total of 50 nasal swap were investigated for HAdV, throughout th PCR technique by using specific primer. The results showed 18 (36.0%) were positive and 32 (64.0%) were negative for HAdVs. The statistical analysis revealed no significant difference between age groups (X2= 0.786 P. value: 0.375). This study summarized the HAdV were the predominant types identified in children less than five years intend to have respiratory tract infection according to clinical symptoms of upper or lower respiratory infections.by using conventional PCR for identify with highly sensitively.

Identification of non-model mammal species using the MinION DNA sequencer from Oxford Nanopore.

The Neotropics harbors the largest species richness of the planet; however, even in well-studied groups, there are potentially hundreds of species that lack a formal description, and likewise, many already described taxa are difficult to identify using morphology. Specifically in small mammals, complex morphological diagnoses have been facilitated by the use of molecular data, particularly from mitochondrial sequences, to obtain accurate species identifications. Obtaining mitochondrial markers implies the use of PCR and specific primers, which are largely absent for non-model organisms. Oxford Nanopore Technologies (ONT) is a new alternative for sequencing the entire mitochondrial genome without the need for specific primers. Only a limited number of studies have employed exclusively ONT long-reads to assemble mitochondrial genomes, and few studies have yet evaluated the usefulness of such reads in multiple non-model organisms. We implemented fieldwork to collect small mammals, including rodents, bats, and marsupials, in five localities in the northern extreme of the Cordillera Central of Colombia. DNA samples were sequenced using the MinION device and Flongle flow cells. Shotgun-sequenced data was used to reconstruct the mitochondrial genome of all the samples. In parallel, using a customized computational pipeline, species-level identifications were obtained based on sequencing raw reads (Whole Genome Sequencing). ONT-based identifications were corroborated using traditional morphological characters and phylogenetic analyses. A total of 24 individuals from 18 species were collected, morphologically identified, and deposited in the biological collection of Universidad EAFIT. Our different computational pipelines were able to reconstruct mitochondrial genomes from exclusively ONT reads. We obtained three new mitochondrial genomes and eight new molecular mitochondrial sequences for six species. Our species identification pipeline was able to obtain accurate species identifications for up to 75% of the individuals in as little as 5 s. Finally, our phylogenetic analyses corroborated the identifications from our automated species identification pipeline and revealed important contributions to the knowledge of the diversity of Neotropical small mammals. This study was able to evaluate different pipelines to reconstruct mitochondrial genomes from non-model organisms, using exclusively ONT reads, benchmarking these protocols on a multi-species dataset. The proposed methodology can be applied by non-expert taxonomists and has the potential to be implemented in real-time, without the need to euthanize the organisms and under field conditions. Therefore, it stands as a relevant tool to help increase the available data for non-model organisms, and the rate at which researchers can characterize life specially in highly biodiverse places as the Neotropics.

Selection of marker systems for determining the allelic composition of high molecular glutenin loci in winter wheat

Aim. Selection of marker systems and conducting multiplex polymerase chain reactions to determine the allelic composition of the Glu-A1, Glu-B1 and Glu-D1 loci, which determine the high quality of flour in common wheat samples. Methods. Polymerase chain reaction (PCR) using specific primers for the Glu-A1, Glu-B1 and Glu-D1 loci of wheat. Results. PCR analysis was performed to detect the insertion at 43 b. p. in the MAR region, in varieties with the Glu-B1al allele, the presence of an amplicon 563 b. p. long was observed, for varieties that carry other alleles of the locus – an amplicon 520 b. p. long. The results on the Glu-D1 locus showed that for № 2, № 3, № 4, № 7, № 8, № 10, № 11, № 19, № 20, № 21 subunits (5 + 10) were identified. Conclusions. With the help of allele-specific primers, the allelic composition of loci of high molecular weight glutenins was determined in 22 lines wheat. Three alleles Glu-A1a, Glu-A1b, Glu-A1c were found in the Glu-A1 locus; in the Glu-B1 locus, only the Glu-B1al allele was detected, there are two alleles at the Glu-D1 locus: Glu-D1a and Glu-D1d (the Glu-D1a allele predominated). As a result of our research, samples of soft wheat (lines № 2, № 7, № 21) were selected, which can be attributed to genotypes with high baking quality of the flour.

First Report of Citrus Leaf Blotch Virus on Nagami Kumquat in Turkey.

Kumquat, known as the smallest of citrus fruits, has seen an increase in the number of commercial orchards in recent years. In May of 2022, a PCR-based survey for citrus virus diseases was conducted in commercial Nagami kumquat (Fortunella margarita (Lour.) Swingle) orchards in the Cukurova region of Turkey. During the survey, five trees with bud union disorder symptoms were found in two different kumquat orchards in the Erdemli district of Mersin province. Young leaf samples from the five trees showing bud union disorder symptoms and two symptomless trees were collected from 6-year-old kumquat. Common citrus viruses and viroids, such as citrus psorosis virus, citrus exocortis viroid, and hop stunt viroid, were not detected in PCR tests performed on all collected samples. The samples were also tested for citrus leaf blotch virus (CLBV; genus Citrivirus, family Betaflexiviridae), a virus associated with a bud union crease in kumquat (Vives et al.2001). Total RNA was isolated from collected leaf samples using the RNeasy Plant Mini Kit (Qiagen, USA). In the RT-PCR assay, two separate specific primer pairs were used to determine the presence of CLBV. The coat protein gene (CP), was amplified with primers KU-18 (5'-TTAAGATTACAGACACGAAGG- 3') and KU-19 (5' CTGTTTTTGAATTTTGCTCG-3'), and the RNA-dependent RNA polymerase gene (RdRp) was amplified with KU-27(5'-GATGCAAGCCAGGATGAATAC-3') and KU-15 (5'-CAGACACTCCAAGACCTTTCC-3') primers (Vives et al. 2002). All of symptomatic samples yielded amplicons of the expected size (456-bp RdRp and 438-bp CP). No amplicons were obtained from symptomless samples. Two PCR products were sequenced to confirm their identity by direct sequencing (GenBank accession no: OQ718432, OQ718433, PP726107, PP726108). Consensus sequences of these two genes showed 96% and 97% nt identity with CP and RdRP genes of the CLBV sequence in GenBank (MT038390, EU857539), respectively. For biological indexing, four RT-PCR-positive samples were used as inoculum sources. Four of Etrog citron (C. medica) and four of Dweet tangor (C. tangerina) seedlings were graft-inoculated for each source, and two seedlings for each cultivar were used as a negative control. Two months later, chlorotic blotching symptoms were observed on Dweet tangor leaves. Six months later, Etrog citron exhibited stem pitting symptoms. None of the negative control seedlings developed any symptoms. RT-PCR was performed to confirm the presence of CLBV in the virus-inoculated seedlings, and amplicons of the expected sizes (456-bp RdRp and 438-bp CP) were obtained. To the best of our knowledge, this is the first report of the CLBV infection in citrus in Turkey. CLBV is thought to have originated from infected grafting material in the country, although it is not known where it may have originated. Further research is needed to determine the distribution of CLBV and its potential to impact commercial citrus production in Turkey.

First Report of Rhizome Rot Caused by Pectobacterium aroidearum on Ginger in China.

Ginger (Zingiber officinale) is a vital commercial crop in China, valued for its medicinal and nutritional properties. From June to September 2023, soft rot symptoms were observed on ginger rhizomes in fields in Shucheng County, Lu'an City, Anhui Province, China (31°24'N, 116°53'E). Brown, water-soaked lesions emerged at the stem base, accompanied by leaf yellowing and wilting. Approximately 20% of the ginger fields in this region (about 530 hectares) displayed soft rot symptoms. Rhizomes from ten randomly diseased ginger plants from three different locations were surface disinfected (Liu et al. 2020). The disinfected rhizomes were ground in sterile water, and serial dilutions of the supernatant were then placed on Luria-Bertani (LB) agar, resulting in the isolation and purification of 432 colonies. Using the primer set 27F/1492R (Weisburg et al. 1991), the 16S rRNA gene was amplified and sequenced. BLASTn analysis indicated that Pectobacterium species were isolated from all three locations, accounting for one-fifth of the total isolates. Three isolates, PA03, PA05, and PA08, were randomly chosen for further analysis. These colonies appeared milky white, round, with a smooth surface and smooth margins. All three isolates were Gram-negative, tested negative for methyl red and catalase, but positive for urease. The 16S rRNA sequences obtained (GenBank accession No. PP935462, PP935465 and PP935466) were 100% identical to Pectobacterium aroidearum strains KNUB-05-21 (LC777355, 1,375/1,375 bp), 3898C (KY446053, 1,418/1,418 bp), and MY11 (MT834965, 1,373/1,373 bp). The icdA, mdh, and proA genes of the isolates were amplified by PCR using specific primer pairs icdA-F/icdA-R, mdh-F/mdh-R, and proA-F/proA-R, respectively (Ma et al. 2007). BLASTn searches showed that the icdA (PP922142), mdh (PP922148), and proA (PP922145) sequences of PA03 had similarities of 99.25%, 98.26%, and 98.01%, respectively, with those of P. aroidearum strain UNEB3 (MH500095, 563/536 bp; MH507329, 522/394 bp; MH507331, 655/702 bp). Similarly, the icdA (PP922143), mdh (PP922149), and proA (PP922146) sequences of PA05 were 99.05%, 99.09%, and 97.18% identical to those of P. aroidearum strain MTL1911110204 (MT013063, 529/552 bp; MT013079, 498/399 bp; MT013095, 717/714 bp). The icdA (PP922144), mdh (PP922150), and proA (PP922147) sequences of PA08 shared 99.25%, 98.27%, and 97.94% similarity with those of P. aroidearum strain CCRM35 (MH500096, 537/537 bp; MH507330, 513/396 bp; MH507332, 630/705 bp). Phylogenetic trees were generated using MEGA11.0 software based on 16S rRNA and concatenated sequences of icdA-mdh-proA (Tamura et al. 2021). The results of phylogentic analysis revealed that the isolates, PA03, PA05, and PA08, clustered with P. aroidearum strains. To test pathogenicity, 10 µl of bacterial suspensions (1×108 CFU/ml) of isolates PA03, PA05, and PA08 were inoculated into the stem base of ginger plants using a sterile syringe, with sterile water used as a control. Each isolate was inoculated into five pots, with three plants per pot. The plants were maintained in a greenhouse at 28°C with 75% relative humidity. Rhizomes inoculated with bacterial suspensions displayed soft rot symptoms resembling those observed in the field seven days after inoculation, while the control group remained free of symptoms. Bacteria were re-isolated from diseased tissues and identified by PCR with specific primer pairs icdA-F/icdA-R, mdh-F/mdh-R, and proA-F/proA-R. The re-isolated strains had the identical sequences as PA03, PA05, and PA08, confirming their pathogenicity and fulfilling Koch's postulates. P. aroidearum is known to cause soft rot in various crops (Barroso et al. 2019; Morase et al. 2020; Tang et al. 2021). To our knowledge, this is the first report of P. aroidearum causing soft rot disease of ginger rhizomes in China. In 2023, soft rot disease led to a 15 to 20% reduction in ginger yield, posing a substantial risk to ginger cultivation in Lu'an city. Identifying this pathogen is crucial for preventing disease outbreaks and developing effective disease management strategies.

The Potential of JWH-133 to Inhibit the TLR4/NF-κB Signaling Pathway in Uterine Ischemia–Reperfusion Injury

In recent years, significant progress has been made in understanding the biological and molecular pathways that regulate the effects of ischemia–reperfusion (I/R) injuries. However, despite these developments, various pharmacological agents are still being tested to either protect against or mitigate the damage caused by the IR’s harmful consequences. JWH133 is a CB2R-selective agonist and belongs to the class of Δ8-tetrahydrocannabinol. The present study aimed to determine the in vivo effect of JWH-133 on uterine IR injury via the TLR4/NF-κB, pathway. Female Wistar albino rats (n = 40) were randomly divided into five groups. Three different doses of JWH-133 (0.2, 1, and 5 mg/kg) were administered to the rats. RNA was isolated from uterine tissue samples, and gene expression was measured by RT-PCR using specific primers. The interaction energies and binding affinities of JWH-133 with IL-1β, IL-6, NF-κB, TLR-4, and TNF-α were calculated through molecular docking analysis. The expression analysis revealed that JWH-133 administration significantly reduced the expression levels of IL-1β, IL-6, NF-κB, TLR-4, and TNF-α (p < 0.05). Notably, in the 1 mg/kg JWH-133 group, all of the gene expression levels decreased significantly (p < 0.05). The molecular docking results showed that JWH-133 formed hydrogen bonds with GLU64 of IL-1β, SER226 of IL-6, and SER62 of TNF-α. This study highlights the molecular binding affinity of JWH-133 and its potential effects on inflammation in IR injury. These results pave the way for future research on its potential as a therapeutic target.

Fall Armyworm Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) in East Nusa Tenggara Province of Indonesia: Genetic Characterization and Strain Detection

<em>Spodoptera frugiperda</em> J.E. Smith (Lepidoptera: Noctuidae) is an invasive pest of corn plants spreading throughout the world, including East Nusa Tenggara (NTT), Indonesia. Despite the wide distribution, there is a lack of information on the strain or genetic diversity of the pest in NTT. Therefore, this study aimed to determine the strain of <em>S. frugiperda</em> from several areas in NTT with a molecular method using <em>cytochrome oxidase subunit 1</em> (<em>COI</em>)<em> </em>and <em>triose phosphate isomerase</em> (<em>Tpi</em>)<em> </em>gene markers. The<em> </em>samples were collected from 3 islands: Timor, Flores, and Sumba. Amplification of the marker genes was carried out using 3 specific primers to identify the strain obtained from samples. Subsequently, polymerase chain reaction (PCR) products were sequenced and the DNA sequences were analyzed using the BioEdit and BLAST programs. Phylogeny analyses were carried out using the MEGA 11 program to verify the strain group of samples with reference isolates from other countries found in GenBank. The PCR results showed product amplicon size 811 bp for the <em>CO1A</em> marker. Based on phylogeny tree analysis using <em>COI </em>marker, <em>S. frugiperda </em>from NTT showed 2 clades, namely corn and rice strains. The characterization results showed that <em>S. frugiperda</em> in NTT comprised 63.6% corn and 36.4% rice strains. <em>COIB </em>classified <em>S. frugiperda</em> from NTT into the h4 haplotype subgroup, while <em>Tpi </em>gene marker was in the corn strain. This study provided valuable information regarding the strain of <em>S. frugiperda</em> in Indonesia to determine the appropriate control strategy.

NDM-5-producing Klebsiella pneumoniae ST258 in a university hospital in Argentina

Abstract Background A drastic increase in carbapenem resistance among Klebsiella pneumoniae isolates occurred during the period 2019–22. Three epidemiological changes could be evidenced: (i) NDM became the predominant carbapenemase; (ii) NDM-5 replaced NDM-1; and (iii) the emergence of NDM-producing K. pneumoniae ST258 (NDM-KpST258). Materials and methods Carbapenem-resistant K. pneumoniae isolates from patients on the ICU of a university hospital of Buenos Aires were studied during the period 2019–22. Identification was performed by MS and susceptibility by the Phoenix system (broth microdilution for colistin). Carbapenemase production was detected phenotypically. Molecular studies included PCR with specific primers and WGS (in some isolates). Results NDM-producing K. pneumoniae was statistically associated with the use of ceftazidime/avibactam between 2019 and April 2021, whereas in the period from May 2021 to December 2022, it seemed to be related to the presence of NDM-5-KpST258. A gradual increase in the number of urease-negative NDM-Kp-ST258 during 2019–22 was observed. The plasmid origin of NDM-5 was supported by its presence on the IncFII incompatibility group plasmid. Conclusions Our study describes the first outbreak of NDM5-KpST258 at an ICU in Argentina, remarkably associated with considerable changes in the carbapenemase epidemiology. The intrinsic characteristics of ST258 may contribute to increased spread of NDM in hospital settings, resembling KPC-2 dissemination.

Direct quadruplex real-time VPCR for simultaneous rapid detection of Pinelliae Rhizoma, Rhizoma Pinelliae Pedatisectae and Rhizoma Typhonii Flagelliformis

Known for its health benefits, Pinelliae Rhizoma is frequently utilized in many botanical dietary supplements and herbal products. However, the substantial demand has led to the frequent adulteration with morphologically similar species, such as Rhizoma Pinelliae Pedatisectae and Rhizoma Typhonii Flagelliformis. Addressing this challenge, a direct quadruplex real-time VPCR approach has been developed in this study. Firstly, a direct lysis protocol has been implemented to facilitate the rapid release of genomic DNA within only 2 min. Secondly, specific primers and probes were designed for Pinellia ternata (Thunb.) Breit., Pinellia pedatisecta Schott and Typhonium flagelliforme (Lodd.) Blume, respectively. A conserved sequence on the ITS was also included as an internal control to mitigate the potential false negatives arising from DNA extraction or DNA amplification errors. Furthermore, the amplification process has been refined through a VPCR heating model, curtailing the detection to a scant 30 min. After a series of optimizations, the developed direct quadruplex VPCR allows for the simultaneous detection of these three plant materials, with each material having a detection sensitivity as low as 1 pg/µL. At last, the method's applicability was validated by using several commercial samples previously identified by Sanger sequencing.

A Multiplex PCR Assay for Simultaneous Detection of Giardia duodenalis, Cryptosporidium parvum, Blastocystis spp. and Enterocytozoon bieneusi in Goats.

Giardia duodenalis, Cryptosporidium parvum, Blastocystis spp. and Enterocytozoon bieneusi are four common zoonotic parasites associated with severe diarrhea and enteric diseases. In this study, we developed a multiplex PCR assay for the simultaneous detection of these four zoonotic protozoans in goat stool samples and assessed its detection efficiency. Specific primers were designed from conserved gene sequences retrieved from GenBank, and the PCR conditions were optimized. Genomic DNA from 130 samples was subjected to both single-target PCR and multiplex PCR. The multiplex PCR assay successfully amplified specific gene fragments (G. duodenalis, 1400 bp; C. parvum, 755 bp; Blastocystis spp., 573 bp; E. bieneusi, 314 bp). The assay sensitivity was ≥102 copies of pathogenic DNA clones with high specificity confirmed by negative results for other intestinal parasites. The detection rates were 23.08% (30/130) for G. duodenalis, 24.62% (32/130) for C. parvum, 41.54% (54/130) for Blastocystis spp., and 12.31% (16/130) for E. bieneusi, matching the single-target PCR results. The sensitivity and predictive values were 100.00%. This multiplex PCR provided a rapid, sensitive, specific, and cost-effective approach for detecting these four parasites. It also provided essential technical support for the rapid detection and epidemiological investigation of G. duodenalis, C. parvum, Blastocystis spp., and E. bieneusi infections in goat fecal samples.

Research Note: Development of a reverse transcriptase recombinase-aided amplification method for detection of Parrot Borna Virus 4

The Parrot Borna virus 4 (PaBV-4) is the primary causative agent of parrot developmental disorder (PDD), leading to symptoms such as bloating, undigested feed in stool, decreased appetite, diarrhea, and weight loss. Its impact on the parrot industry has been significant, therefore, it is imperative to develop a rapid detection method for PaBV-4. The detection of PaBV-4 was achieved through the development of an RT-RAA assay, which involved the design of specific probes and primers targeting the N gene. This method allows for detection at 41°C within 30 min and has a minimum detection threshold of 8.56 × 101 copies/μL. The RT-RAA method demonstrated specific detection of PaBV-4 without any cross-reactivity observed with H5N6, H7N9, H9N2 avian influenza virus, newcastle disease virus (NDV), avian infectious bronchitis virus (IBV) and Parrot Borna virus 2 (PaBV-2). The coefficient of variation for the 3 repeatability experiments was below 10%. Tissue samples from 28 suspected cases of PaBV related deaths in parrots were analyzed using both RT-RAA and RT-qPCR methods. The sensitivity and specificity of both methods were 100%, demonstrating perfect agreement between them as indicated by a kappa value of 1. In conclusion, this study created a RT-RAA method for PaBV-4 detection successfully.

Genetic Diversity and Relationship of Native Phalaenopsis Orchids: A Case Study of Indonesian Archipelago

The native Phalaenopsis is valuable germplasm for future orchid breeding programs and for its conservation because it provides many beneficial traits or genes. This study aimed to determine and analyze the molecular diversity and phylogeny of Indonesian native Phalaenopsis by a DNA barcoding (matK) marker. A total of 19 samples of Phalaenopsis orchids were used in this study. All leaf samples of orchid were extracted and purified using a commercial DNA isolation kit from Geneaid Biotech Ltd., Taiwan (GP100). The DNAs were then amplified by specific matK primers: Forward (5’-CGTACAGTACTTTTGTGTTTACGAG-3’) and Reverse (5’-ACCCAGTCCATCTGGAAATCTTGGTTC-3’). The DNA targets or products (matK) were purified and sequenced by the Sanger-bidirectional method at 1st Base Ltd., Malaysia. Before further analysis, the matK sequences of Phalaenopsis were edited, reconstructed, and aligned with the assistance of Clustal W in the MEGA 11 software. Its genetic diversity was determined using the nucleotide diversity index (π%), and the phylogenetic analysis was performed using the maximum likelihood (ML) method with a statistical bootstrap. The phylogenetic relationship was also assessed using principal component analysis (PCA). Based on this marker, the native Phalaenopsis has a high genetic diversity (π% = 1.70). In addition, the phylogenetic analysis revealed that this germplasm was separated into seven clades, where P. pantherina has the closest relation to P. cornu-cervi and P. gigantea. Conversely, the highest genetic distance was to P. amabilis from South Kalimantan and to P. celebensis from Sulawesi, at a coefficient divergence of 0.084. Our findings provide an essential foundation for supporting future orchid breeding practices, including conservation, on local and global scales.

First Report of Fusarium avenaceum Causing Root Rot of Raspberry (Rubus corchorifolius) in China.

Raspberry (Rubus corchorifolius) plants hold historical, economic, and medicinal importance in China (Yang et al. 2022). Raspberries are cultivated to generate income for local farmers in Lintao County, Dingxi City, Gansu Province. However, farmers encountered challenges due to raspberry plants exhibiting root rot disease, resulting in plant death. During a thorough field survey conducted in June 2022, symptoms ranging from leaf yellowing and wilting to necrotic lesions and root rots were observed, where approximately 30% of raspberry plants were affected. Five diseased and healthy plants were collected from the farmers' fields in Lintao (35.53oN, 103.84oE) for pathogen identification. Symptomatic and asymptomatic root tissues were surface sterilized with 75% ethanol for 30 s and 3% NaOCl for 5 min, followed by three rinses in sterile water. Small pieces (0.5 × 0.5 cm) were cut and incubated on potato dextrose agar (PDA) plates at 25°C for 7-10 days. Twenty-two pure Fusarium isolates, which displayed four distinct colony groups morphologically, were obtained. Pathogenicity tests on isolates RB10, RB1, RB30, and RB23, representing each colony group, revealed that RB10 exhibited symptoms similar to those observed in the field. The RB10 strain produced yellowish-white to greyish-white colonies on PDA and was then cultured in a carboxymethylcellulose (CMC) broth for enhanced conidia production (Zhang et al. 2020). Macroconidia were sickle-shaped or slightly curved, with three to five septa (19.2 to 38.5 x 3.1 to 5.8 µm, n =40). Microconidia were oval to ellipsoidal, non-septate or featuring 1 to 2 septa (4.8 to 10.5 x 2.1 to 5.2 µm, n=20). These morphological features indicated the isolate was similar to Fusarium avenaceum (Leslie and Summerell, 2006). For further identification of the strains, genomic regions (ITS-rDNA, TEF-1α, and RPB2) were amplified and sequenced using specific primers ITS1/ITS4, EF-1/EF-2, and 5f2/7cr, respectively (O'Donnell et al. 2010; Uwaremwe et al. 2021; Zarrin et al. 2016). PCR BLASTn queries of NCBI GenBank revealed a 99.8% (522 bp), 99.4% (355 bp) and 99.6% (985 bp) homology with F. avenaceum (MZ724839.1, MN271631.1, and MK185026.1), respectively. Sequences were deposited in GenBank (ITS, OR735571; TEF-1α, PP216660; RPB2, PP857820). One-year-old raspberry seedlings were planted in pots with a sterile soil mix (2:2:1 v/v ratio of soil, peat, and vermiculite) under controlled greenhouse conditions (23-26°C, 16h light/8h dark). A month post-planting, taproots were wounded in six pots and inoculated with 20 ml of conidia suspension (106 conidia/ml), while the other six pots were maintained as controls. After 14 days, RB10-infected plants showed symptoms similar to field observations, while controls remained healthy. The experiment was conducted twice, and re-isolation confirmed both the pathogenicity and identity of the pathogen. In the concatenated phylogenetic tree of ITS, TEF-1α and RPB2, strain RB10 was clustered with the F. avenaceum representative strains KG502, KG431 and F094. Studies revealed F. avenaceum varied pathogenicity across plants (Bugingo, 2022; Moparthi et al. 2020& 2024; Yli-Mattila et al. 2018), and it has been reported to induce raspberry fruit rot (Wang et al. 2017). However, no previous reports linked this fungus to raspberry root rot. This report is crucial for understanding the impact of root rot disease on raspberry cultivation and developing effective management strategies.

Development and application of a quantitative real-time PCR method for detection of Decapod iridescent virus 1.

As a newly discovered virus, Decapoda iridovirus 1 (DIV1) can cause a mortality rate of up to 100% in crustaceans, leading to huge economic losses. At present, there is no effective prevention and control measures for this disease. In the present study, the specific primers targeting highly conserved regions of MCP gene were designed, and then a quantitative real-time PCR method was established. The results indicate that DIV1 quantitative real-time PCR established has good specificity and does not cross react with other pathogens including white spot syndrome virus (WSSV), infectious subcutaneous and hematopoietic necrosis virus (IHHNV) and Vibrio parahaemolyticus induced acute hepatopancreatic necrosis disease (VpAHPND). The real-time PCR was capable of detecting DIV1 DNA at a minimum concentration of 10 copies/μL within 34 cycles. The method has good repeatability, with intra group and inter group coefficients of variation both less than 2%. Thirty-two clinical samples were assessed using both the real-time PCR and conventional PCR. The results shown real-time PCR we established are more sensitive than conventional PCR. In conclusion, this method has strong specificity, stable repeatability, and high sensitivity, providing technical support for clinical diagnosis, epidemiology investigation and monitoring of DIV1.

The value of Mentzer index in the diagnosis of children thalassemia minor

ABSTRACT Objective: To explore the effect of the Mentzer Index (MI) in screening for children thalassemia (TT) minor. Methods: We determined the MI cutoff value of diagnosing TT minor in children by utilizing a receiver operating characteristic (ROC) curve. Samples with MI values below this threshold, and no detectable gene mutations or IDA were further analyzed using Sanger sequencing or specific primer GAP-PCR. Results: In the differential diagnosis of silent α-thalassemia (α-TT) and IDA, the area under the ROC curve (AUC) for the Mentzer index (MI) was 0.561. The most Youden index indicates an MI cutoff value of 16.833, with a sensitivity of 1.000 and a specificity of 0.278. For distinguishing α-TT minor from IDA, the AUC was 0.909, with an MI cutoff value of 12.24, a sensitivity of 0.875, and a specificity of 0.778. When distinguishing β-TT minor from IDA, the AUC was 0.907, with an MI cutoff value of 11.82, a sensitivity of 0.917, and a specificity of 0.768. Utilizing these MI cutoff values, three cases of rare TT genotype were identified, including a previously unreported heterozygous mutation of IVS-II-55 (T > G) in the alpha2 (αIVS-II-55(T > G)/αα). Conclusion: The diagnostic precision of MI is limited when differentiating between silent α-TT and IDA, but it is higher when distinguishing between α-TT minor, β-TT minor, and IDA. This study established a cutoff value for MI in our laboratory, providing reliable data for screening for TT minor and discovering rare TT genotypes among children in the local area.

First report of Rhizoctonia blight caused by Rhizoctonia solani on Chinese cabbage (Brassica rapa subsp. chinensis) in India.

During January and February 2021, foliar blight symptoms were observed on the leaves of Chinese cabbage (Pak choi) at Lembucherra research farm, College of Agriculture, Tripura, India. The incidence of disease symptoms ranged from 5 to 10% of the plants observed in the field. The symptomatic leaves showed grayish colored water-soaked lesions with an irreguar shape and size. A total of 10 symptomatic leaves (1 leaf per plant) from Chinese cabbage infected plant were sampled, surface decontaminated with 1% NaOCl, washed twice in sterile water, plated on 2% water agar, and incubated at 25 ± 2°C. Hyphal tips from mycelium of 7-day old culture (2 isolates from two different plants) with right-angled branching were transferred to potato dextrose agar (PDA) media (SRL, India). Cream or light brown hyphae that branched at right angles, with septa near the point of the origin of hyphae, and a slight constriction at the base of the branch) were visible under a microscope. Olive-brown sclerotia were observed after 5 days of incubation. Multiple nuclei per cell were visible after staining with 4', 6-diamidino-2-phenylindole (Estandarte et al. 2016). Based on morphological characteristics (Parmeter et al. 1970) the isolates TP36 and TP37 were identified as Rhizoctonia solani. The internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were amplified with ITS1& ITS4 (White et al. 1990) and (GAPDH F-5'- CAAGGAGAACCCAGGTGTTAAG-3' and GAPDH R- 5'-GGCGTCGAAGATAGAAGAGTGT-3') respectively for both isolates and sequenced (accession #. PP458158, PP458159, PP425343, PP425344). BLASTn analysis showed 99.26%( 668/673 nt) to 99.46% (659/664 nt) identity with R. solani sequences (GenBank MG397062.1 and KX674524.1) for ITS and 98.42% (552/562 nt) to 100% 540/540 nt)identity with R. solani sequences (GenBank HQ425709.1 and CP102644.1) for GAPDH. Isolates TP36 and TP37 were deposited in the Indian Type Culture Collection (ITCC), New Delhi as R. solani (nos. 9154 and 9319, respectively). Both isolates were amplified using (anastomosis group) AG1 subgroup specific primers (Matsumoto 2002; Prashantha et al. 2021) to identify their AG. The presence of a 265 bp amplicon for both isolates suggested that they belong to AG1-IA. A multilocus analysis of R. solani isolates from different host plants with concatenated sequences ITS and GAPH showed that TP36 and TP37 are closely related to rice isolate RS107. A pathogenicity test on five plants per treatment was conducted and repeated twice on one month old Chinese cabbage plants (hybrid, TOKITA, India) grown under glasshouse conditions in a sterilized mixture of soil and sand (3:1) at 27-28oC during January 2024 at ICAR-IARI, New Delhi. R. solani isolates TP36 and TP37 were grown on PDA and plants were inoculated by placing single sclerotia of 10-day old colony on different plant parts and covering it with moist cotton. After 7 day, typical lesions of R. solani infection were visible. No symptoms were observed on the control plants. The fungus was reisolated from the inoculated plants and identified as R. solani based on morphology. R. solani has previously been reported to cause disease on some members of Brassicaceae in different countries (Budge et al. 2009; Hua et al. 2014). Based on literature available this is the first report of R. solani infecting Chinese cabbage in India.

Standardization and application of ARMS TaqMan real-time PCR for screening of folate metabolism genes in Han Chinese.

Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time-consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time-saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS-PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real-time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real-time PCR in SNP genotyping was 1ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG+GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real-time PCR is a reliable SNP genotyping method in folate metabolism genes.

First report of potato virus H infecting tomato (Solanum lycopersicum) in China.

Potato virus H (PVH), belonging to the genus Carlavirus in the family Betaflexiviridae, was initially discovered in potato plants in Inner Mongolia, China (Li et al., 2013). Subsequently, it was documented to infect pepino, a perennial shrub of the Solanaceae family like potatoes (Abouelnasr et al., 2014). Tomato (Solanum lycopersicum L.), a major global crop, faces threats from various plant viruses. In an open field survey in Yunnan, China during July 2023, tomatoes (cultivar: Liangsi) showed typical virus symptoms: leaf yellowing, curling, mottling, and fruit with abnormal shape and color. Eleven symptomatic tomato samples were collected for high-throughput sequencing to identify the potential pathogen. RNA sequencing libraries were prepared using the TruSeq RNA sample prep kit (Illumina, San Diego, CA, USA), followed by RNA-seq sequencing on an Illumina HiSeq4000 platform (LC Sciences, USA). Approximately 77,928,560 paired-end reads (150-bp each) were generated. After quality control, 75,808,296 reads were retained and subjected to de novo assembly using Trinity (version 2.8.5). The assembled contigs, ranging from 198 nt to 15865 nt, were used as queries to search against the NCBI non-redundant protein sequence database (NR) or nucleotide sequence database (NT) to detect the potential pathogens using BLASTx and BLASTn program with a cutoff e-value of 10-5. As a consequence, certain contigs were assigned to 3 plant viruses, including PVH (the highest RdRp blastx identity to UAD82396.1: 97.8%), Capsicum chlorosis virus (CaCV, the highest RdRp blastx identity to APQ31267.1: 98.4%), and southern tomato virus (STV, the highest CP-RdRp fusion protein blastx identity to QOW17541.1: 99.74%). The presence of the identified 3 viruses was subsequently screened in the 11 tomato samples originally collected from the corresponding field. Notably, the specific detection primers for the PVH genome was designed from the newly assembled PVH genome (Forward primer: 5'- ATAGTTGTGCACTGTGTGCCTG-3'; Reverse primer: 5'-GCTTAAGGTTCTTAGCGTATTC-3'), targeting ~1.1kb. Consequently, PVH was detected in 3 out of 11 samples: 2 leaf samples and 1 fruit sample, with one leaf sample showing a single infection. The complete genome sequence of PVH in tomatoes (PVH-tomato) was successfully obtained by assembling nine overlapping regions spanning the entire PVH-tomato genome, following the RT-PCR and the 5' RACE and 3' RACE approaches, and deposited in NCBI nucleotide database with accession number OR397130.1Phylogenetic analysis based on the full genome sequences of PVH-tomato and other publicly available PVH isolates revealed that PVH-tomato was closely related to a PVH isolate found in potatoes in Yunnan (blastn similarity: 97.76%) (Fig. S1A). To test PVH-tomato infectivity and pathogenicity, four healthy Nicotiana benthamiana and four healthy tomato plants were mechanically inoculated with PVH-infected leaf sap; controls used sap from healthy plants. Three weeks post-inoculation, all N. benthamiana (4/4) and three tomato plants (3/4) were PVH-positive by RT-PCR. Symptoms were milder in N. benthamiana, and only two tomato plants (2/4) showed leaf curling. No PVH was detected in control samples (Figure S1B, S1C). Sanger sequencing confirmed the amplicons' expected length of 1093 bp. Previously, PVH was documented only in potato and pepino. This is the first report of tomatoes as natural PVH hosts and PVH infecting N. benthamiana under lab conditions.

Fusarium species and assessments of mycotoxin (deoxynivalenol), in wheat seeds from different regions of Türkiye

Wheat cultivation is important in Turkish agriculture, which ranks 10th among international wheat producers, and is an important wheat exporter, particularly to Europe. Fusarium-related threats, such as Fusarium Head Blight (FHB) and Fusarium Crown and Root Rots (FCR, FRR), and related mycotoxin seed contamination, jeopardize product quality. This study analysed 65 wheat seed samples for presence of Fusarium species, from cultivars of Triticum aestivum (bread wheat) and T. durum (durum wheat) collected from seven regions of Türkiye. PCR with specific primers, and phylogenetic analyses of TEF1-α segments, discriminated Fusarium species. Levels of the mycotoxin deoxynivalenol (DON) in flour samples were also evaluated. Out of 195 Fusarium isolates, the prominent species included F. graminearum (32% of isolates), F. proliferatum (16%), F. avenaceum (11%), F. clavum (11%), and F. verticillioides (7%). Less frequently isolated species were F. oxysporum (6%), F. acuminatum (3%), F. ramigenum (3%), F. culmorum (3%), F. poae (2%), F. sambucinum (2%), F. tricinctum (2%), Fusarium sp. FTSC12 (2%), F. andiyazi (1%), and F. equiseti, F. incarnatum, and F. fasciculatum (each 0.5%). Five of the 65 samples tested positive for DON, with two exceeding the European Commission threshold for mycotoxin contamination; one bread wheat from the Black Sea region, known for its annual rainfall, and a durum wheat sample from southeastern Anatolia, which had the highest detected DON level of 1730 μg kg-1. Among these samples F. graminearum was the predominant species. As F. andiyazi and F. ramigenum are not normally associated with wheat plants, a pathogenicity test was conducted with two isolates of each of these species, revealing no pathogenicity on the durum wheat cultivar ‘San Carlo’. These results provide a basis for managing fungal threats and mycotoxin contamination, safeguarding the quality of wheat grain as an essential agricultural product.