Search Results (27)

(1) Background: Centella asiatica (L.) is a long-lived medicinal plant traditionally recognized for its wound-healing and anti-inflammatory properties. Despite the growing demand for diverse C. asiatica species in Korea, studies on genetic diversity remain limited. (2) Methods: Genome assembly data of C. asiatica from the NCBI database were utilized to develop genomic SSR markers. Genetic diversity and population structure were examined in 30 Korean native C. asiatica accessions using 90 SSR markers. (3) Results: Whole-genome sequencing revealed 376,751 SSR loci, from which 127,528 primer pairs were designed. Among 160 randomly selected primers, 90 showed consistent amplification and displayed high levels of polymorphism. Genetic analyses revealed that the MAF ranged from 0.15 to 1.00 (mean 0.55), the NA ranged from 1 to 15 (mean 5.6), the Ho ranged from 0.00 to 1.00 (mean 0.17), and the PIC values ranged from 0.00 to 0.88 (mean 0.52). Clustering analysis with 90 SSR markers revealed three clusters, whereas population structure analysis indicated two populations among the C. asiatica accessions. Furthermore, two minimum marker sets with five marker combinations were identified and proved useful to differentiate all C. asiatica accessions. (4) Conclusions: The newly developed SSR markers for C. asiatica hold promise for facilitating research endeavors pertaining to variety identification, genetic mapping, and marker-assisted selection. Full article

The teak defoliator, Hyblaea puera, native to South Asia and Southeast Asia (e.g., India, Laos, and Myanmar), has recently caused frequent outbreaks in mangrove forests across Guangdong, Guangxi, and other regions of China. Its larvae feed extensively on the leaves of Avicennia marina, severely threatening local mangrove ecosystems. However, accurate morphological identification of H. puera across its eggs, larvae, and pupae remains challenging. Therefore, the development of rapid molecular detection methods is essential for effective pest identification and monitoring, thereby supporting timely management interventions. In this study, mitochondrial protein-coding genes (PCGs) were analyzed from H. puera and related species were analyzed. Sliding window analysis was conducted to estimate nucleotide diversity (Pi), leading to the selection of the cytochrome c oxidase subunit I (COI) gene as the optimal target. Species-specific primers were designed based on the H. puera COI sequence, and two molecular detection assays—SS-PCR and LAMP—were developed. Both assays exhibited high specificity, stability, and sensitivity, successfully amplifying target fragments from H. puera across all tested geographic populations and different developmental stages. The limit of detection of the SS-PCR method was 83 fg/µL DNA, while that of the LAMP method reached 8.3 fg/µL DNA. The newly developed assays offer reliable and robust tools: the SS-PCR method is suitable for precise, large-scale detection in laboratory settings, whereas the LAMP assay is preferable for rapid, field-based detection of H. puera. These methods contribute to the early detection and effective management of H. puera populations, thereby safeguarding mangrove ecosystems. Full article

Point-of-care diagnostics are revolutionizing the detection of plant pathogens by enabling rapid, on-site identification without the need for specialized laboratories. One of the tools used for this purpose is loop-mediated isothermal amplification (LAMP). LAMP is a powerful molecular technique increasingly used in pathogen control for its rapid, sensitive, and specific detection of plant pathogens. The aim of this study was the development of a novel, easy-to-use portable colorimetric LAMP (cLAMP) assay that could be used by inexperienced personnel for the detection of the pathogen Clavibacter michiganensis. The assay was combined with a newly constructed device in which LAMP can be performed in 30 min. Initially, a new set of LAMP primers targeting the micA gene was designed and evaluated the sensitivity (100 fg/reaction) and specificity of the assay. Next, the limit of detection (LoD) of two different commercial LAMP kits was compared with common laboratory detection techniques (DAS-ELISA, immunofluorescence, quantitative PCR, and PCR) using the same samples. Additionally, the LoD of the developed cLAMP assay was evaluated in bacterial suspensions and plant extracts spiked with C. michiganensis and validated the effect on the LoD of plant extracts from different tomato varieties. Lastly, its efficacy for C. michiganensis detection was assessed in experimentally inoculated tomato seedlings. The developed method for C. michiganensis detection can be used as a reliable tool for the early detection of the pathogen for field-based applications by untrained personnel. Full article

Aspergillus oryzae and Aspergillus flavus are closely related species within the Aspergillus section Flavi, sharing approximately 99.5% genomic similarity. Despite this similarity, they differ markedly in their ability to produce aflatoxin, a carcinogenic mycotoxin synthesized by the aflatoxin biosynthesis gene cluster (ABGC). Species and strains included within section Flavi display diverse deletion patterns in the ABGC at the sequence level. In this study, we performed an in-depth comparative analysis of the ABGC of 30 strains belonging to section Flavi, including isolates obtained from nuruk. The analysis revealed that A. oryzae exhibits distinct large-scale or locus-specific deletions in the ABGC compared to other related species. Based on these unique deletion patterns, we designed four primer sets to distinguish A. oryzae from A. flavus by comparing the sizes of PCR amplicons. Application of these primer sets to nuruk-derived isolates enabled successful species differentiation with 92% accuracy. To further validate this method, in silico PCR analysis was conducted using publicly available genomes of A. oryzae (116) and A. flavus (482), confirming that the developed biomarkers could consistently distinguish between the two close species. The primer sets are expected to serve as a rapid, accurate, and practical method for distinguishing A. oryzae from A. flavus. Full article

Fusarium spp. represent a critical threat to maize production and food safety due to their mycotoxin production. This study introduces a refined molecular identification protocol integrating four genomic regions—ITS1, IGS, TEF-1α, and β-TUB—for robust species differentiation of Fusarium spp. isolates from post-harvest maize in Bulgaria. The protocol enhances species resolution, especially for closely related taxa within the Fusarium fujikuroi species complex (FFSC). A newly optimized multiplex PCR strategy was developed using three primer sets, each designed to co-amplify a specific pair of toxigenic genes: fum6/fum8, tri5/tri6, and tri5/zea2. Although all five genes were analyzed, they were detected through separate two-target reactions, not in a single multiplex tube. Among 17 identified isolates, F. proliferatum (52.9%) dominated, followed by F. verticillioides, F. oxysporum, F. fujikuroi, and F. subglutinans. All isolates harbored at least one toxin biosynthesis gene, with 18% co-harboring genes for both fumonisins and zearalenone. This dual-protocol approach enhances diagnostic precision and supports targeted mycotoxin risk management strategies. Full article

Despite numerous studies on haemosporidians in wild birds from Brazil, the presence of other vector-borne agents (VBA) such as Anaplasma spp., Bartonella spp., and Onchocercidae filariids in avian hosts remains largely unknown. The low occurrence of these VBAs might be due to the low sensitivity of traditional molecular techniques. The microfluidic real-time PCR assay, known for its high sensitivity, has emerged as a promising method to detect and study the occurrence and diversity of VBAs in both arthropod vectors and vertebrate hosts. To validate previously and standardize newly designed microfluidic real-time PCR protocols, selected positive avian blood DNA samples for Anaplasma spp., Bartonella spp., haemosporidians, and filariids were used. The molecular occurrence rates for the selected VBAs were 18.2% for Anaplasma spp., 0.36% for Bartonella spp., 6.2% for Plasmodium spp., 4.7% for Haemoproteus spp., and 6.5% for Onchocercidae filariids. The Plasmodium spp. cytB sequence detected in a Volatinia jacarina clustered with Plasmodium tejerai, whereas the Haemoproteus spp. cytB sequence detected in a Columbina squamata clustered with Haemoproteus columbae. While Onchocercidae filariid cox-1 sequences were detected in specimens of Ramphocelus carbo, Turdus amaurocalinus and Synallaxis albilora grouped with Aproctella spp., one sequence detected in R. carbo was ancestral to the clade comprising Splendidofilaria spp. and Eufilaria spp. High-throughput microfluidic real-time PCR assay can be used for screening VBAs in avian hosts from South America, but new primers/probe sets should be designed for VBA genotypes present in Brazil. Full article

Duck hepatitis B virus (DHBV) is widely prevalent in global ducks and has been identified in Chinese geese with a high prevalence; the available detection techniques are time-consuming and require sophisticated equipment. In this study, an assay combining multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) was developed for the efficient and rapid detection of DHBV. The primary reaction condition of the MIRA assay for DHBV detection was 10 min at 38 °C without a temperature cycler. Combined with the LFD assay, the complete procedure of the newly developed MIRA assay for DHBV detection required only 15 min, which is about one-fourth of the reaction time for routine polymerase chain reaction assay. And electrophoresis and gel imaging equipment were not required for detection and to read the results. Furthermore, the detection limit of MIRA was 45.6 copies per reaction, which is approximately 10 times lower than that of a routine polymerase chain reaction assay. The primer set and probe had much simpler designs than loop-mediated isothermal amplification, and they were only specific to DHBV, with no cross-reactivity with duck hepatitis A virus subtype 1 and duck hepatitis A virus subtype 3, goose parvovirus, duck enteritis virus, duck circovirus, or Riemerella anatipestifer. In this study, we offer a simple, fast, and accurate assay method to identify DHBV in clinical serum samples of ducks and geese, which would be suitable for widespread application in field clinics. Full article

Lavers are typically consumed in dried or seasoned forms. However, commercially processed lavers can lead to seafood fraud because it is impossible to authenticate the original species based on morphological characteristics alone. In this study, we developed a capillary electrophoresis-based multiplex polymerase chain reaction (PCR) to authenticate six different laver species. The species-specific primer sets to target the chloroplast rbcL or rbcS genes were newly designed. We successfully established both singleplex and multiplex conditions, which resulted in specific amplicons for each species (N. dentata, 274 bp; N. yezoensis, 211 bp; N. seriata, 195 bp; N. tenera, 169 bp; N. haitanensis, 127 bp; P. suborbiculata, 117 bp). Moreover, the assays were sensitive enough to detect DNA ranging from 10 to 0.1 pg of DNA. The optimized capillary electrophoresis-based multiplex PCR was successfully applied to 40 commercial laver products. In addition to detecting the laver species as stated on the commercial label, the assay discovered cases where less expensive species were mixed in. With its advantageous properties, such as short amplicon size, high specificity, and superior sensitivity, this assay could be used for the authentication of the six laver species. Full article

Papaver somniferum L. is cultivated for its edible seeds and for the production of alkaloids. A serious problem in seed trade and processing is the intentional mixing of excellent food-quality seeds with non-food-grade-quality seeds. Tracking the correct or illegitimate handling of seeds requires an efficient method for discrimination and individualization of poppy varieties. As in human and animal forensics, DNA variable regions containing short tandem repeats (STRs) located either in non-coding DNA or in gene sequences (EST-STRs) are preferred markers for discrimination between genotypes. Primers designed for 10 poppy EST-STR loci not analyzed so far were tested for their discriminatory ability on a set of 23 related P. somniferum L. genotypes. Thirty-three EST-STR alleles were identified together. Their polymorphic information content (PIC) values were in the range of 0.175–0.649. The PI value varied in the range of 0.140–0.669, and the cumulative PI was 1.2 × 10⁻⁵. PI_sibs values varied between 0.436 and 0.820 and the cumulative value was lower (5.0 × 10⁻³). All analyzed genotypes were distinguished mutually, each with its own unique EST-STR profile. These newly developed EST-STR markers more effectively discriminated P. somniferum L. genotypes, even those genotypes whose DNA profiles were previously identical. Full article

Both sterile and non-sterile pharmaceutical products, which include antiseptics, have been recalled due to Burkholderia cepacia complex (BCC) contamination. Therefore, minimizing the frequency of outbreaks may be conducive to the development of a quick and sensitive approach that can distinguish between live and dead loads of BCC. We have assessed an exo probe-based recombinase polymerase amplification (RPA) with 10 µM propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in various concentrations of antiseptics (i.e., chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions) after 24 h. The optimized assay conducted using a set of primer–probes targeting gbpT was performed at 40 °C for 20 min and shows a detection limit of 10 pg/µL of genomic DNA from B. cenocepacia J2315, equivalent to 10⁴ colony-forming units (CFU/mL). The specificity of a newly designed primer and probe was 80% (20 negatives out of 25). The readings for total cells (i.e., without PMAxx) from 200 µg/mL CHX using PMAxx-RPA exo assay was 310 relative fluorescence units (RFU), compared to 129 RFU with PMAxx (i.e., live cells). Furthermore, in 50–500 µg/mL BZK-treated cells, a difference in the detection rate was observed between the PMAxx-RPA exo assay in live cells (130.4–459.3 RFU) and total cells (207.82–684.5 RFU). This study shows that the PMAxx-RPA exo assay appears to be a valid tool for the simple, rapid and presumptive detection of live BCC cells in antiseptics, thereby ensuring the quality and safety of pharmaceutical products. Full article

The Human Immunodeficiency Virus (HIV) is a highly morphic, retrovirus that rapidly evolves through mutation as well as recombination. Because of the immunocompromised status in HIV patients, there is often a higher chance of acquiring different secondary infections followed by liver cirrhosis, hepatitis B & C, and HIV-associated nephropathy. The current study was conducted to see the prevalence of secondary infections, hematological and biochemical markers for liver and renal associated diseases, and to detect the envelope gene (GP41) in newly diagnosed HIV patients. A total of 37 samples were collected from HIV-positive patients registered in different hospital settings under the National AIDS control program. The collected samples were processed for hepatitis B, hepatitis C, hematological analysis, and biochemical analysis. To identify the envelope gene in newly diagnosed HIV patients, polymerase chain reaction (PCR) was performed using four gene-specific primers. The HIV infections were seen more in male as compared to females. A significant decrease in complete blood count was observed in HIV patients when compared to healthy individuals. There was a significant increase in aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine observed in HIV patients. No significant difference was observed in alkaline phosphatase (ALP), total bilirubin, and albumin levels when compared to healthy control. Anemia was observed in 59.4% of HIV patients. A total of three (8.1%) patients were found to be co-infected with hepatitis B and one (2.7 %) was co-infected with hepatitis C. Out of these 37 tested samples, a total of four showed the successful amplification of the envelope gene. This study provides platform for the health care facilitators to regularly monitor the signs, symptoms and clinical biomarkers of HIV-associated infections to prevent toxicity at an early stage to improve the quality of life (QoL) and minimize the mortality rate in HIV patients. Envelope gene mutating frequently results in drug resistance, and thus future research on polymorphism analysis will reveal points of substitutions to improve drug designing. Full article

Listeria monocytogenes is a major human foodborne pathogen responsible for listeriosis. The virulence factor Internalin A (inlA) has a key role in the invasion of L. monocytogenes into the human intestinal epithelium, and the presence of premature stop-codons (PMSC) mutations in the inlA gene sequence is correlated with attenuated virulence. The inlA sequencing process is carried out by dividing the gene into three sections which are then reassembled to obtain the full gene. The primers available however were only able to entirely amplify the lineage II isolates. In this study, we present a set of new primers which allow inlA sequencing of isolates belonging to both lineages, since lineage I isolates are the ones most frequently associated to clinical cases. Using newly designed primers, we assessed the presence of inlA PMSCs in food, food processing environments and clinical isolates. Full article

Porcine epidemic diarrhea virus (PEDV) can cause severe infectious porcine epidemic diarrhea (PED) and infect different ages of pigs, resulting in sickness and death among suckling pigs. For PEDV detection, finding an effective and rapid method is a priority. In this study, we established an effective reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for PEDV detection. Three sets of primers, specific for eight different sequences of the PEDV N gene, were designed in this study. The optimized RT-LAMP amplification program was as follows: 59 min at 61.9 °C and 3 min at 80 °C. The RT-LAMP results were confirmed with the addition of SYBR Green I fluorescence dye and with the detection of a ladder-like band by conventional gel electrophoresis analysis, which demonstrated a significant agreement between the two methods. The LOD of PEDV by RT-LAMP was 0.0001 ng/μL. Compared with RT-LAMP, the traditional RT-PCR method is 100-fold less sensitive. The RT-LAMP results had no cross-reaction with porcine parvovirus (PPV), porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), rotavirus (RV), transmissible gastroenteritis virus (TGEV) and porcine reproductive and respiratory syndrome virus (PRRSV). Consequently, the newly developed RT-LAMP method could provide an accurate and reliable tool for PEDV diagnosis. Full article

Enzootic bovine leukosis (EBL) is a disease caused by bovine leukemia virus (BLV); only a small percentage of BLV-infected cattle develop EBL and present with B-cell lymphosarcoma. There is no vaccine against BLV, treatment for EBL, or method for predicting the possibility of EBL onset, thus making EBL control difficult. Herein, to explore biomarkers for EBL in milk, we examined the mRNA profiles of small extracellular vesicles (sEVs) in milk from four BLV-uninfected and four EBL cattle by microarray analysis. It was revealed that 14 mRNAs were encapsulated in significantly higher quantities, and these mRNAs were therefore selected as biomarker candidates. Primers for these mRNAs were designed, and nine primer sets were available for quantitative real-time PCR. Nine mRNAs were evaluated for their availability as biomarkers for EBL using sEVs from newly-collected milk of 7 uninfected and 10 EBL cattle. The quantities of eight mRNAs (TMEM156, SRGN, CXCL8, DEFB4A, FABP5, LAPTM5, LGALS1, and VIM) were significantly higher in milk sEVs of EBL cattle than in those of uninfected cattle. Therefore, our findings indicate that these eight mRNAs in milk sEVs can be used as potential EBL biomarkers with combination use, although single mRNA use is not enough. Consequently, cattle at risk of EBL onset can be identified by monitoring the fluctuation in quantities of these mRNAs in milk before they develop EBL. Full article

Honey is a widely used natural product and the price of honey from Apis cerana (ACH) and A. dorsata (ADH) is several times more expensive than the one from A. mellifera (AMH), thus there are increasing fraud issues reported in the market by mislabeling or mixing honeys with different entomological origins. In this study, three species-specific primers, targeting the NADH dehydrogenase 2 (ND2) region of honeybee mitochondrial DNA, were designed and tested to distinguish the entomological origin of ACH, ADH, and AMH. Molecular analysis showed that each primer set can specifically detect the ND2 region from the targeted honeybee DNA, but not from the others. The amplicon size for A. cerana, A. dorsata and A. mellifera were 224, 302, and 377 bp, respectively. Importantly, each primer set also specifically produced amplicons with expected size from the DNA prepared from honey samples with different entomological origins. The PCR adulteration test allowed detection of 1% of AMH in the mixture with either ACH or ADH. Furthermore, real-time PCR and melting curve analysis indicated the possible discrimination of origin of honey samples. Therefore, we provide the newly developed PCR-based method that can be used to determine the entomological origin of the three kinds of honey. Full article