Gene-specific Primers Research Articles

Environmental detection and genetic profiling of Acanthamoeba in high-risk medical facilities.

Environmental detection and genetic profiling of Acanthamoeba in high-risk medical facilities.

Fusobacterium nucleatum is not significantly present in eutopic endometrium from patients with minimal-mild and moderate-severe endometriosis.

Fusobacterium nucleatum is not significantly present in eutopic endometrium from patients with minimal-mild and moderate-severe endometriosis.

Mycobacterium marinum flexor tenosynovitis of the hand diagnosed by tissue polymerase chain reaction: a case report

Mycobacterium marinum, a non-tuberculous mycobacterium, is commonly found in contaminated water or fish and can lead to deep infections of the hand. Identification of this bacterium is challenging, and traditional microbial culture and identification methods may result in delayed diagnosis and treatment. Tissue polymerase chain reaction is a diagnostic method that directly amplifies genus-specific primers from infected tissues obtained during surgery to identify mycobacterial species, and this approach provides results faster than conventional culture methods. We report a case of refractory infectious flexor tenosynovitis of the hand, in which the causative organism had not been identified for several months through smears, culture, and detection tests. Through extensive debridement and tissue polymerase chain reaction as an identification test, the patient was diagnosed with M. marinum infection and successfully treated.

First Report of 'Candidatus Phytoplasma trifolii'-related strain Association with Little Leaf and Declining Disease of Pulasan (Nephelium mutabile Blume) in Kerala, India

Pulasan (Nephelium mutabile Blume; family Sapindaceae) is a tropical fruit known for its high orilagin, ascorbic acid and thiamine content, known for its potent antioxidant and anti-inflammatory properties. Native to Southeast Asia, pulasan is extensively cultivated in Malaysia, Thailand, Indonesia, Sri Lanka, India and Philippines (Afzaal et al., 2023). In India, pulasan is cultivated in tropics, where it is cherished as a homestead crop. A recent survey conducted in September 2024 at Pangode village (8°45'23.22"N 76°58'48.68"E), Thiruvananthapuram, Kerala, showed little leaf symptoms along with drying of side branches along with stunted growth in pulasan trees (variety P15). Only two pulasan trees were recorded with symptoms out of 20 trees examined in the campus. To investigate the molecular identity of the phytoplasma, leaf samples from two symptomatic and asymptomatic trees were collected and subjected to total DNA extraction by DNA extraction kit (Qiazen). The extracted DNA was amplified using nested PCR assays with universal primers P1/Tint (Smart et al., 1996) followed by 3Fwd/3Rev (Manimekalai et al., 2010) specific for the 16S rRNA gene and tuf gene-specific primer pairs fTufl/rTufl (Schneider et al., 1997). The PCR assays yielded amplicons of ~1.3 kb and ~1.1 kb for the 16S rRNA and tuf genes, respectively, from all the symptomatic leaf samples, whereas no amplification was observed from any asymptomatic samples. One sequence of each gene of pulasan phytoplasma strain (PK-1) were deposited into the GenBank database [(Acc Nos. PQ865399 (16S rRNA gene) and PQ877893 (tuf gene)]. BLASTn analysis of the both gene sequences of the PK strains revealed a 99.62% sequence identity with eggplant little leaf (Acc. No. MW273757; 16S rRNA gene) and 100% sequence similarity with Cucurbita pepo phytoplasma (Acc. No. KX358589; tuf gene) belongs to the clover proliferation phytoplasma subgroup. Phylogenetic analysis using MEGA 11 employing the Maximum likelihood method, demonstrated that the PK phytoplasma strains clustered within the 16SrVI-D subgroup-related strains. The virtual RFLP pattern generated for the 16S rRNA gene of PK phytoplasma strain through pDRAW analysis was found identical to the reference pattern of 16VI-D subgroup (Periwinkle little leaf phytoplasma; GenBank Acc. No. AF228053). Sequence comparison analysis confirmed the presence of a 'Candidatus Phytoplasma trifolii'-related strain associated with pulasan showing little leaf disease and stunting in India. While, phytoplasma associations have been reported in various fruit species globally (Fiore et al., 2018; Mitra et al., 2023), this study represents the first documented case of a phytoplasma disease infecting pulasan worldwide. Notably, 'Ca. P. trifolii' (16SrVI-D subgroup) has previously been identified associated with several economically important agricultural and horticultural crops worldwide (Zhao and Wei, 2020; Rao, 2021). The results of this study revealed that pulasan trees serve as an alternative natural reservoir for the ‘Candidatus Phytoplasma trifolii’ (16SrVI-D) phytoplasma strain, may potentially facilitate its dissemination to other crops through insect vectors. Considering the significant phytosanitary concerns, epidemiological factors and economic impact of pulasan in Southeast Asia for its global export potential, the detection of phytoplasma-associated disease in pulasan is noteworthy as it not only poses a potential threat to cultivation and productivity but may also have broader implications for regional trade and biosecurity protocols.

Co-Occurrence of Beauvericin and Fumonisin Producing Ability of Fusarium Strains Isolated from Crop Plants in Hungary

Beauvericin (BEA) is an emerging mycotoxin with wide-ranging bioactivity (antimicrobial and insecticide), making it a potential target for drug and pesticide development. BEA primarily produced by Beauveria, Isaria, and Fusarium species. The BEA-producing abilities of a collection of 100 Fusarium strains isolated from maize were tested using a gene-specific primer (Beas_1, Beas_2) by PCR. Among all, 23 were found to have the beauvericin synthetase (BEAS) gene sequence, which is responsible for the production of BEA. Fusarium proliferatum (6) and F. verticillioides (14) strains were producing the highest BEA concentrations. The toxin-producing ability of the strains was investigated in small bioreactors. Parallel with BEA, the most frequent Fusarium toxins such as deoxynivalenol (DON), T2, HT-2, zearalenone (ZEA), fumonisin B1 (FB1), and fumonisin B2 (FB2) were also measured. Only FB1 and FB2 were observed above the detection limit, the coexistence of the FBs and BEA was measured in high concentration. In all BEA-producing strains, the FBs production could be detected. The highest BEA concentration was 3131 mg/kg, and the highest FB1 and FB2 concentrations were 4393 mg/kg and 1390 mg/kg, respectively. In the present study, the gene sequences responsible for the production of BEA in F. verticilloides isolates have not only been detected but also demonstrated with UHPLC-ESI-MS/MS to be capable of biosynthesis. From the phylogenic analysis of the BEAS gene sequences, the assumption could be made that the ability to produce BEA was conferred via horizontal gene transfer.

Heterorhabditis bacteriophora Extracellular Vesicles Alter the Innate Immune Signaling in Drosophila melanogaster

Background: Heterorhabditis bacteriophora entomopathogenic nematodes are commonly used in agricultural practices for the biological control of insect pests. These parasites are also used in basic research for unveiling the molecular basis of nematode parasitism in relation to the insect anti-nematode response. We have recently shown that H. bacteriophora excreted–secreted products reduce the expression of the antimicrobial peptide gene Diptericin in Drosophila melanogaster, which increases fly mortality due to enhanced propagation of the mutualistic bacteria Photorhabdus luminescens. However, the effect of entomopathogenic nematode extracellular vesicles (EVs) on the insect host defense remains unknown. Methods: Here, we injected adult flies with H. bacteriophora EVs and used quantitative RT-PCR together with gene-specific primers to analyze the activity of immune-related signaling pathways. Results: We found that H. bacteriophora EVs are lethal to Drosophila melanogaster, and they downregulate the expression of Attacin, Cecropin, and Prophenoloxidase 3 in adult flies. Conclusions: These findings build on previous knowledge and strengthen the notion that H. bacteriophora entomopathogenic nematodes release a variety of effector molecules to modify the insect’s innate immune signaling. This information is important because it contributes toward clarifying the molecular interplay between entomopathogenic nematode components and the host’s innate immune system.

Comparison Of Expression Levels Of Keratin 19 (Krt19) and Transmembrane Serine Protease 4 (Tmprss4) Genes in SU.86.86 and BxPC-3 Pancreatic Cancer Cell Lines

In this study, the expression levels of Keratin 19 (KRT 19) and Transmembrane serine protease 4 (TMPRSS4) genes in metastatic pancreatic cancer cell line SU.86.86 and nonmetastatic pancreatic cancer cell line BxPC3 cells were been determined and the capacity of these two genes to be metastatic biomarkers in pancreatic cancer is aimed to be evaluated. Genomic DNA was isolated from BxPC3 and Su8686 cell lines, which we replicated in culture medium, and then cDNA was obtained. By using gene-specific primers, expression levels of KRT 19 and TMPRSS4 genes were determined at the transcriptional level by real time PCR method. At the end of this study TMPRSS4 gene expression was found to be 1.31-fold decreased and KRT19 gene expression was found to be 1234-fold increased in the metastatic pancreatic cancer cell line SU8686. TMPRSS4 gene expression was found to be 2.38-fold decreased and KRT19 gene expression was found to be 183-fold increased in the non-metastatic BXPC3 cell line. In pancreatic cancers, where survival is very low, most of the patients are in the metastatic process at the time of diagnosis. For this reason, there is an urgent need for metastatic biomarkers that can provide information about the prognosis of the disease. In this study, the expression of the KRT19 gene at the transcriptional level was found to be quite high in the metastatic pancreatic cancer cell line. For this reason, it is thought that studies with large patient groups may be beneficial as a metastatic biomarker candidate in pancreatic cancer.

Therapeutic Insights Into a Case-Control Approach to B-cell Lymphoma 3 (BCL3)-Encoded Protein by Exploring Immune Modulation and Clinical Strategies in Oral Carcinomas.

Oral cancer ranks as one of the most prevalent malignancies worldwide. While the role of B-cell lymphoma 3 (BCL3)-encoded protein as an oncogene has been explored in other epithelial cancer types, its specific contribution to oral carcinoma (OC) remains insufficiently investigated. This study investigated the expression of BCL3 patterns in two OC subtypes and examined its influence on immune-related mechanisms. A cross-sectional evaluation based on a case-control study was conducted from September 2022 to January 2023, enrolling 100 participants, of whom 77 were patients diagnosed with OC and 23 were healthy control participants. Two subtypes, squamous cell carcinoma (SCC) and mucoepidermoid carcinoma (MEC), were examined. RNA was drawn from both tissue and peripheral blood samples using the QIAamp Blood RNA Kit (#51104, Qiagen, Hilden, Germany). Subsequently, BCL3 expression was quantified using real-time PCR (RT-qPCR) with gene-specific primers. Statistical analysis was performedusing one-way ANOVA via IBM SPSS Statistics for Windows, Version 20 (Released 2011; IBM Corp., Armonk, New York), with a threshold of p < 0.05. The highest BCL3 expression was seen in SCC samples (2.91 ± 0.62), followed by MEC samples (1.87 ± 0.58), while healthy controls showed baseline levels of expression (0.94 ± 0.49). The elevated expression of BCL3 is highly correlated with the advanced stage of the tumour, reduced immune infiltration, and multi-modal treatment requirements. Consequently, BCL3 appeared to put a modulatory force on the immunity environment of tumours in OC, particularly in SCC cases.

Evaluation of native Bacillus thuringiensis strains possessing nematicidal specific cry genes against Meloidogyne incognita.

Plant-parasitic nematodes, including root-knot nematodes, are phyto-parasites that cause significant crop damage and economic losses. Bacillus thuringiensis (Bt), which produces nematicidal toxins, is extensively used to combat nematode infestations in agricultural and horticultural crops. This research assessed the efficacy of native Bt strains as a biocontrol agents against the root-knot nematode Meloidogyne incognita. Twenty native Bt strains were evaluated for the presence of nematicidal cry genes using PCR. Eight strains, namely Bt1, Bt5, Bt6, Bt7, Bt17, Bt19, Bt23, and Bt24, exhibited the presence of nematicidal cry genes, specifically cry5, app6, cry12, cry13, cry14, cry21, xpp55, cry31, cry73, and cry40, as determined by gene-specific primers. The in vitro effectiveness of the Bt strains was assessed against M. incognita using a cavity block test, revealing that the Bt strains, namely Bt7 and Bt19, impeded the hatching of M. incognita eggs and were deadly to nematode larvae (J2 stage). SEM analysis of spore-crystal mixtures of Bt isolates revealed different crystal shapes that confirmed the nematicidal activity. Pot experiments revealed that the Bt7 and Bt19 strains are the most efficacious biological agents, exhibiting superior nematicidal activity in Brinjal and Tomato. The molecular characterization of the most virulent Bt strains, namely Bt-7 and Bt-19, using 16S rDNA sequencing, validated their molecular identification as Bacillus thuringiensis.

Registration of two peanut mutants TifGP‐8 and TifGP‐9 with reduced expression of peanut allergens Ara h 1.02 and Ara h 2.02

Abstract Peanut (Arachis hypogaea L.) is a nutritious food source; however, it can cause serious allergic response among sensitized children and adults. Sixteen peanut proteins have been identified as allergens. Among those, vicilin protein Ara h 1 and conglutinin Ara h 2 are considered major peanut allergens due to the high frequency of recognition by IgE from allergic patients. Reducing the accumulation of Ara h 1 and Ara h 2 in peanut could potentially alleviate the severity of peanut allergic reactions. In order to identify allergen reduced lines, an ethyl methane sulfonate mutagenized peanut population was screened with gene specific primers by Targeting Induced Local Lesions in Genomes (TILLING) approach. TifGP‐8 (Reg. no. GS‐13, PI 701083) is a mutant line carrying a transitional C to T mutation in Ara h 1.02 which resulted in a premature stop codon and truncation in protein synthesis. TifGP‐9 (Reg. no. GS‐14, PI 701084) carries a G to A transition at the start codon of Ara h 2.02 resulting in a knockout of protein expression. Two cleaved amplified polymorphic sequence markers targeted at the mutation loci were developed to identify homozygous lines among progenies. Release of these allergen mutants will provide valuable genetic stocks for breeding reduced allergen peanuts.

Abstract 3039: Elucidating hsa_circ_0001038 and circRNA_400029 circular RNAs mechanisms in response to splicing factor 3 inhibitor in cervical cancer cells

Abstract Cervical cancer (CCa) is the fourth leading cause of cancer-related death among women worldwide. Around 90% of CCa cases are reported in low- and middle-income countries (LMICs), with Sub-Saharan Africa (SSA) bearing over 80% of the global burden. CircRNAs hsa_circ_0001038 and circRNA_400029 have been implicated in cervical cancer. Splicing factor 3 key a key factor in circRNA generation. However, mechanisms of these ceRNAs in CCa is poorly understood. This study aimed at elucidating hsa_circ_0001038 and circRNA_400029 mechanisms in response to splicing factor 3 inhibitor, theophylline in CCa. Using bioinformatics, CircAtlas, the study identified miRNA binding sites for hsa_circ_0001038, selecting miR-205-5p for further analysis due to its tumor-suppressive role. Similarly, miRTarBase predicted miR-16-5p as a key interacting miRNA for circRNA_400029. The downstream gene targets of these miRNAs were explored, focusing on those associated with CCa. Cell cycle FACS analysis and apoptosis assay were used to study cell biological response of theophylline to CCa cells SiHa and C33A. Furthermore, gene specific primers of hsa_circ_0001038, circRNA_400029 and SRSF3 were used to study gene expression in drug treated and control groups. Interestingly, ceRNA network was constructed, showing that one gene can be targeted by more than one miRNA. Additionally, pathway analysis using KEGG, STRING, and Reactome highlighted key signaling pathways, such as the HPV E5-EGFR-RAS-ERK and HPV E6-Notch pathways, which are implicated in cell proliferation, survival, and oncogenesis in CCa. Cell cycle analysis revealed G2/M arrest in response to theophylline treatment, while there was no significant cytotoxic effects of the drug, as revealed by apoptosis FACS analysis. hsa_circ_0001038, circRNA_400029 and SRSF3 were downregulated by theophylline treatment. Understanding circular RNAs and their interaction with splicing factors holds potential translational and clinical applications. Citation Format: Amahle Nyalambisa, Zodwa Dlamini, Rahaba Makgotso Marima. Elucidating hsa_circ_0001038 and circRNA_400029 circular RNAs mechanisms in response to splicing factor 3 inhibitor in cervical cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3039.

Specific detection of genetically modified potatoes containing asparagine synthetase-1 and polyphenol oxidase 5 genes derived from potato

ABSTRACT Several genetically modified (GM) potatoes have been developed by introducing endogenous genes derived from potatoes, such as asparagine synthetase-1 (Asn1) and polyphenol oxidase 5 (Ppo5), to improve quality. Therefore, it is difficult to distinguish between GM and non-GM potatoes. In this study, we developed a sequence-specific polymerase chain reaction (PCR) detection method to identify innate and inserted genes. We designed four Asn1 gene-specific primers and eight construct-specific detection primers to evaluate GM potatoes (E12, X17, and Y9) and non-GM crops. Consequently, PCR products corresponding to the original endogenous potato genes and the inserted genes were clearly distinguished and simultaneously identified. In addition, the PCR method demonstrated sufficient sensitivity to identify GM content at levels as low as 0.5%. Thus, this study offers an effective detection method for monitoring or screening approved and unapproved GM potato events using Asn1 and Ppo5 transgenes in foods and feeds.

Multiplex qPCR Assay for HIV-1 Proviral DNA Detection and Subtype Characterization: Exploiting Quenching of Multiple Fluorophores with a Single Quencher Operating in Trans

Accurate detection and quantification of HIV-1 proviral DNA are critical for effective patient monitoring and therapeutic decision-making. In this study, we developed a multiplexed quantitative PCR (qPCR) assay designed to detect HIV-1 proviral DNA, determine viral subtype, specifically identifying the predominant subtype C and validate assay performance using an internal control. Gene-specific primers were engineered by appending an 8-base biotag followed by a common 18-base sequence at the 5′ end, enabling the simultaneous amplification of multiple target sequences. Fluorescent probes labeled with FAM, SUN/VIC, and Cy5 were employed for detection, and a novel strategy involving quenching of labeled probes in trans was implemented to enhance assay flexibility and cost-effectiveness compared to conventional cis-quenched probes. The assay was initially optimized using synthetic linear double-stranded DNA templates representing the HIV-1 gag region, while externally added human chromosomal DNA served as a control for PCR inhibition. Validation was performed on a panel of 11 clinical samples previously analyzed for drug resistance mutations. Results indicated robust amplification of HIV-1 proviral DNA, accurate subtype determination, and reliable internal control performance, with profiles closely matching those obtained by gold standard sequencing-based assays. One sample exhibited PCR inhibition, underscoring the need for internal control monitoring. Overall, the multiplexed qPCR assay provides a sensitive, specific, and efficient tool for comprehensive HIV-1 reservoir quantification and molecular epidemiological studies, potentially informing improved clinical management and personalized treatment strategies. Furthermore, this novel methodology significantly reduces reagent costs and processing time while maintaining high sensitivity, making it ideal for routine clinical and research applications.

Systematic follow-up investigation of NSP seroreactors and in-contact cattle and buffaloes for foot-and-mouth disease virus using probang sampling

BackgroundFoot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals. In India, the FMD Control Program has been ongoing for the last two decades. A 3AB3 nonstructural protein (NSP)-based indirect ELISA test is used for population serosurveys to differentiate between infected and vaccinated animals (DIVA). In the present study, a systematic follow-up investigation of the NSP seroreactors and in-contact bovines was carried out from rural cohorts as well as an organized farm in Haryana, India to identify the carrier or neoteric animals. No FMD outbreak was reported from Haryana, a Northern state of India in 2022 and NSP reactivity has also consistently been under 10% for the last five years (2018–2022).ResultsBovines from ten villages of district Hisar, Haryana, demonstrated 5.3% (20/377) [cattle (11.3%; 12/106) and buffaloes (3.0%; 8/271)] FMDV 3AB3 NSP reactivity. Out of those 20 NSP reactors, nine months later, two buffaloes were randomly screened. Both were found negative for NSP reactivity as well as for FMDV in oropharyngeal fluid (OPF) by reverse transcription-multiplex polymerase chain reaction (RT-mPCR) using 1D/2B gene-specific primers. Further screening was done in a herd of regularly vaccinated cattle (n = 11) of an organized farm with no history of FMD outbreaks for more than a decade. All the susceptible animals were vaccinated with FMD + Haemorrhagic septicemia + Black Quarter combined oil adjuvanted vaccine. An NSP reactivity of 36.7% (4/11) in cattle calves 2–4 months after vaccination indicated either the exposure of animals to FMDV or the presence of residual NSPs in the vaccine. None of the OPF samples collected twice from these cattle at intervals of 36–44 days were found to be positive for FMDV with RT-mPCR. The observed NSP seropositivity could be linked to either false positive reactions or evidence of past exposure and virus elimination during OPF sampling. Nearly all animals exhibited protective antibody titers (≥ log10 1.65) against the structural proteins of FMDV serotypes O, A, and Asia-1 by Solid Phase Competitive ELISA (SPCE) indicating the effectiveness of vaccination.ConclusionThe present study provided a preliminary follow-up investigation to assess the status of NSP seroreactors to establish the circulation of FMDV in the animal population, if any, so that the effectiveness of the ongoing vaccination program could be assessed and potential disease-free zones could be identified.

Molecular Detection and Antiserum Preparation of Begomoviruses Infecting Zucchini Squash in Iraq

Abstract Begomoviruses infecting zucchini squash were investigated. Leaf samples were collected from zucchini squash growing areas in Baghdad (Jhadryaa and Yusufiyah), Babylon (Jibela and Mahmudiyah) and Diyala (Khan Bani Saad) Provinces. Samples were screened for the presence of begomoviruses using polymerase chain reaction (PCR) and Deng genus specific primers. Sixteen out of 40 samples were begomovirus positive. Sequence analysis confirmed the detection of Tomato leaf curl Palampur virus (TLCPALV) Begomovirus solanumpalampurense, Squash leaf curl virus (SLCuV) B. cucurbitapeponis and Tomato yellow leaf curl virus (TYLCV) B. coheni in zucchini squash and Datura stramonium samples. Co-infections of B. solanumpalampurense, and B. cucurbitapeponis, B. solanumpalampurense and B. coheni were detected in most samples suggesting begomovirus mixed infections are common in the investigated zucchini squash fields. Datura stramonium can be an alternatives host to B. solanumpalampurense and B. coheni. Total protein extracted from begomovirus infected samples were injected into lab rats to prepare begomovirus specific antibodies. The resultant polyclonal antibodies were labeled using horseradish peroxidase and enzyme linked immune assay (ELISA) was performed. Antibodies prepared could detect begomovirus in infected samples scoring 1.247 highest absorbance value at 405 nm compared to 1.198 of slandered TYLCV antigen. Proteins extracted from begomovirus co-infected tissues can be used to immunize rats for the production of beganovirus specific polyclonal antibodies.

Molecular Epidemiology and Genotypic Diversity of Theileria annulata among Bovines Based Upon Tams1 Gene in India.

Bovine theileriosis, caused by Theileria annulata, presents a significant economic challenge to India's livestock industry. This study investigates the molecular epidemiology of T. annulata in bovine populations from Haryana, India, focusing on the genetic diversity of the Tams1 gene (785bp). The prevalence of T. annulata was evaluated in 800 blood samples using microscopy and PCR techniques, specifically targeting SSU rRNA and Tams1 genes. Genetic diversity among T. annulata isolates was analyzed through DNA sequencing and phylogenetic studies. Additionally, the impact of various risk factors on T. annulata infection was assessed. Microscopy revealed that 29.25% (117/400) of cattle and 2.75% (11/400) of buffalo were detected positive for Theileria spp. in Giemsa-stained blood smears. The Theileria genus-specific primers amplified a 1098bp fragment of the SSU rRNA gene in 39.5% (158/400) cattle and 6% (24/400) buffalo. The T. annulata-specific PCR targeting the partial Tams1 gene revealed a prevalence rate of 37% (148/400) in cattle and 3.5% (14/400) in buffalo. A genetic analysis of the Tams1 gene in 389 sequences, including 14 T. annulata isolates from Haryana (8 from cattle, 6 from buffaloes), revealed significant variation within Indian T. annulata parasites. Analysis of Tams1 gene sequences (389 from 16 countries) revealed 35 haplotypes globally, with four identified from the 14 isolates sequenced in this study. Nucleotide homology among 14 northern Indian isolates ranged from 89.46-100%, compared to a broader range of 78.42-100% when global sequences were included. Among 35 haplotypes, Hap_1 is the most dominant and shows geographic clustering. Globally, low genetic distance (Fst < 0.15) and high gene flow (Nm > 1) were observed among the five populations (South Asia, East Asia, West Asia, Europe, and Africa), suggesting minimal genetic differentiation among T. annulata populations. Negative values in Tajima' s D (-1.21941) and Li's F (-2.97801) tests suggest recent population expansion. Risk factors such as age, sex, and host species are significantly associated with T. annulata infection. This study offers comprehensive insights into T. annulata genetic diversity, population structure, and haplotype networks using the Tams1 gene.

Identification and Genome Characterization of Begomovirus and Satellite Molecules Associated with Lettuce (Lactuca sativa L.) Leaf Curl Disease.

Lettuce (Lactuca sativa L.) plants showing leaf curl and vein enation symptoms were found in Yunnan province, China. PCR detection with genus-specific primers revealed that symptomatic lettuce plants were infected with Begomovirus. The full-length viral component and satellite molecules were obtained by RCA, restriction enzyme digestion, PCR, cloning and DNA sequencing. A viral component (YN-2023-WJ) and three satellite molecules (YN-2023-WJ-alpha1, YN-2023-WJ-alpha2 and YN-2023-WJ-beta) were obtained from diseased lettuce plants. YN-2023-WJ exhibited the highest nt identity at 97.1% with pepper leaf curl Yunnan virus isolated from cigar plants. YN-2023-WJ-beta displayed the highest nt identity at 93.9% with tomato leaf curl China betasatellite. YN-2023-WJ-alpha1 showed the highest nt identity at 94.7% with ageratum yellow vein alphasatellite. YN-2023-WJ-alpha2 shared the highest nt identity at 75.6% with gossypium mustelinum symptomless alphasatellite and vernonia yellow vein Fujian alphasatellite. Based on the threshold for the classification of Begomovirus, Betasatellite and Alphasatellite, YN-2023-WJ was designated as a new isolate of PepLCYnV, YN-2023-WJ-beta as a new isolate of ToLCCNB and YN-2023-WJ-alpha1 as a new member of AYVA, whereas YN-2023-WJ-alpha2 was identified as a new geminialphasatellite species, for which the name pepper leaf curl Yunnan alphasatellite (PepLCYnA) is proposed. To the best of our knowledge, this is the first report of L. sativa L. infection by PepLCYnV associated with ToLCCNB, AYVA and PepLCYnA, and L. sativa L. is a new host plant of Begomovirus.

Molecular cloning and heterologous expression of lipase gene from Pseudomonas aeruginosa in Escherichia coli.

Molecular cloning and heterologous expression of lipase gene from Pseudomonas aeruginosa in Escherichia coli.

Selecting Competent Reverse Transcription Strategies to Maximise Biodiversity Recovery With eRNA Metabarcoding.

Both environmental DNA (eDNA) and environmental RNA (eRNA) have been widely adopted for biodiversity assessment. While eDNA often persists longer in environments, eRNA offers a more current view of biological activities. In eRNA metabarcoding, extracted eRNA is reverse transcribed into complementary DNA (cDNA) for metabarcoding. However, the efficacy of various reverse transcription strategies has not been evaluated. Here we compared the biodiversity recovery efficiency of three strategies: random priming with hexamers, oligo(dT) priming and taxa-specific priming using Mifish-U for fish in both high- and low-biodiversity regions. Our results demonstrate that reverse transcription strategies significantly impact biodiversity recovery. Random priming consistently detected the highest number of taxa in both low- and high-biodiversity regions. In low-biodiversity areas, oligo(dT) performed comparably to random hexamers; however, in high-biodiversity regions, random hexamers outperformed oligo(dT), particularly in recovering rare taxa. While taxa-specific priming was comparable to the other strategies for high-abundance taxa, it was less effective for rare taxa, thus limiting its utility for comprehensive biodiversity assessment. These differences are largely due to the multiple binding sites for random hexamers compared to the fewer or absent sites with oligo(dT) and taxa-specific primers under high eRNA degradation. Combining random hexamers and oligo(dT) significantly improved taxa recovery, especially for low-abundance species, supporting its best practice in eukaryotes. For prokaryotes or genes lacking polyadenylation, random priming is favoured over taxa- or gene-specific priming. Collectively, these findings underscore the critical importance of selecting appropriate reverse transcription strategies in eRNA metabarcoding, with significant implications for effective biodiversity monitoring and conservation efforts.

Identification and characterization of Flowering Locus D in Indian bean [Lablab purpureus (L.) Sweet

The transition from vegetative to floral development is a critical process regulated by a variety of complex internal and external factors. This study aims to characterize the Flowering Locus D (FLD) gene in relation to the floral induction pathway and examine its expression profiles under different photoperiodic conditions in Indian bean (Lablab purpureus (L.) Sweet). The LprFLD gene was PCR-amplified and sequenced using gene-specific primers for both photosensitive (GNIB-22, GP-189) and photoinsensitive (GNIB-21) varieties of Indian bean. Exon prediction, phylogenetic analysis, and in silico domain prediction were performed, followed by gene expression analysis using qRT-PCR for contrasting phenotypes. The complete LprFLD gene sequence was characterized, containing five exons and showing significant similarity to GmFLD and VuFLD. Phylogenetic analysis of FLD homologs from 27 taxa revealed the closest evolutionary relationships among members of the Phaseoleae tribe, including Lablab purpureus, Vignaumbellata, Vigna angularis, Vignaradiata, Vigna unguiculata, Phaseolus vulgaris, Glycine max, Glycinesoja, and Cajanus cajan. In silico domain prediction identified two conserved domains, SWIRM and amino oxidase, in the FLD homologs of Indian bean, soybean, cowpea, Arabidopsis and rice. Gene expression analysis showed that the relative expression of LprFLD in trifoliate leaves corresponded with flowering in both photosensitive and photoinsensitive genotypes under long-day conditions. Overall, the expression level of LprFLD was lower in trifoliate leaves and shoot tips. This is the first report on the characterization of the LprFLD locus in Indian bean. Phylogenetic analysis confirmed its close evolutionary relationship with other Phaseoleae species. qRT-PCR results suggest that the elevated expression of LprFLD in trifoliate leaves coincides with flowering time in both photosensitive and photoinsensitive genotypes under long-day conditions, indicating that other loci may also regulate floral transition under varying photoperiods in Indian bean.