Development and verification of new reference plasmid (pUC_GM-SB) for the DNA-based detection of genetically modified sugar beet H7-1.

The sugar beet is the second-largest sugar-producing crop. Genetically modified (GM) sugar beet, which have herbicide-resistant, have been developed to increase production and comprise over 90% of the market share. This study describes qualitative and quantitative PCR methods for the GM sugar beet H7-1 with reference plasmid (pUC_GM-SB) containing an endogenous gene (GS2) and an event-specific gene for H7-1 that served as a positive control for PCR. The detection limit of qualitative PCR was approximately 10 copies of the reference plasmid and 0.05% in spiked samples. In the case of quantitative PCR, the detection limit was five copies of the reference plasmid. Regarding repeatability, the standard deviation and relative standard deviation were found to range from 0.11 to 0.24 and from 0.23% to 0.99%, respectively. This study provides food safety assurance for imported GM sugar beet H7-1 using the reference plasmid and supports efficient detection methods. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-024-01572-6.

Recombinase Polymerase Amplification assay for detection of the British root-knot nematode, Meloidogyne artiellia.

Recombinase polymerase amplification (RPA) is an isothermal in vitro nucleic acid amplification technique that has been adopted for simple, robust, rapid, reliable diagnostics of nematodes. In this study, the real-time RPA assay and RPA assay combined with lateral flow dipsticks (LF-RPA) have been developed targeting the ITS rRNA gene of the British root-knot nematode, Meloidogyne artiellia. The assay provided specific and rapid detection of this root-knot nematode species from crude nematode extracts without a DNA extraction step with a sensitivity of 0.125 second-stage juvenile (J2) specimen per a reaction tube for real-time RPA during 11 min and a sensitivity of 0.5 J2 specimens per a reaction tube for LF-RPA during 25 min. The RPA assays were validated with a wide range of non-target root-knot nematodes. The LF-RPA assay has great potential for nematode diagnostics in the laboratory having minimal available equipment.

Effects of Black and White Mulch on Mycelial Growth of Tuber Species in Korean Field Environment.

Truffles, belonging to the genus Tuber, are ectomycorrhizal (ECM) fungi that form underground ascocarps and primarily establish symbiosis with oaks and hazels. The cultivation of Tuber spp. involves transplanting inoculated seedlings that have formed ectomycorrhiza with Tuber species, with mulching being effective for truffle cultivation. In this study, we investigated the effects of mulching on the mycelial growth of four Tuber species (T. himalayense, T. koreanum, T. melanosporum, and T. borchii) in the Korean natural environment, highlighting the potential for Korea as a truffle cultivation site. We developed and tested species-specific primers for quantifying the soil mycelial biomass of Tuber spp. by qRT-PCR, determined the superior mulch color for mycelial growth, and identified the Tuber species exhibiting the highest growth rate in the Korean field environment. Our results demonstrated that white mulch significantly enhanced mycelial growth in Tuber species than black mulch, likely owing to its ability to maintain low soil temperatures, control weeds, and improve host plant growth. Among the Tuber species, T. himalayense showed the greatest growth potential in the Korean natural environment. Additionally, a significant and positive correlation was observed between the mycelial biomass of Tuber species and the growth of inoculated seedlings, as measured by the total stem length and the number of leaves, thereby indicating the importance of symbiosis between ECM fungi and host plants. This study provides valuable insights into truffle cultivation in Korea and highlights the potential of using white mulch to promote mycelial growth, thereby contributing essential data for understanding the appropriate environmental conditions for Tuber spp. cultivation in Korea. Further study is needed to assess the long-term impact of mulching and to explore the effectiveness of other mulching materials.

Existence of rare actinobacterial forms in the Indian sector of Southern Ocean: 16 S rRNA based metabarcoding study.

The significance of the Southern Ocean (SO) as a sink of atmospheric CO2 and other greenhouse gases is well established. Earlier studies have highlighted the role of microbes in various SO ecosystem processes. However, the diversity and role of actinobacteria in the Indian sector of SO (ISO) water and sediments are unknown. This study aimed to analyze the diversity of actinobacteria in water and sediment samples of SO based on amplicon microbiome analyses. The taxonomic analysis identified a total number of 27 phyla of which Proteobacteria (40.2%), Actinobacteria (13.6%), and Firmicutes (8.7%) were found to be dominant. The comparative study of water and sediment samples revealed the dominance of different actinobacteria in water and sediments. While the order Streptomycetales was dominant in the water samples, Micrococcales was found to be dominant in the sediment samples. The genus level analysis found the presence of eight and seventeen genera in the sediment and water samples, respectively. The genus Streptomyces, Saccharopolyspora, Nocardioides, Sva0996 marine group, and Mycobacterium were seen both in sediment and water samples. Marmoricola, Ilumatobacter, and Glaciihabitans were observed only in sediment samples whereas Rhodococcus, Corynebacterium, Micrococcus, Turicella, Pseudonocardia, Bifidobacterium, Nesterenkonia, Collinsella, Knoellia, Cadidatus, Actinomarina, Libanicoccus and Cutibacterium were noticed exclusively in water samples. Our study also emphasizes the need for further detailed study to understand the links between actinobacterial diversity and their ecological functions in the ISO. The available metabarcoding data paves the way for future research in cultivable forms of novel and rare Actinobacteria for their bioprospecting applications.

New mitogenomes of Runcinidae and Facelinidae: two understudied heterobranch families (Mollusca: Gastropoda).

Here, we present the mitochondrial sequences of two sea slugs (Heterobranchia): Runcina aurata and Facelina auriculata, the latter being the type species of the family. The mitochondrial genomes are 14,282 and 14,171bp in length, respectively, with a complete set of 13 PCGs, 2 rRNAs, and 22 tRNAs. None of the mitogenomes show gene reorganization, keeping the standard mitogenomic structure of Heterobranchia. Nucleotide composition differs significantly between them, with R. aurata showing the most AT-rich mitogenome (25.7% GC content) reported to date in Heterobranchia, and F. auriculata showing a rich GC content (35%) compared with other heterobranch mitochondrial genomes.

Streamlined whole-genome genotyping through NGS-enhanced thermal asymmetric interlaced (TAIL)-PCR.

Whole-genome genotyping (WGG) stands as a pivotal element in genomic-assisted plant breeding. Nevertheless, sequencing-based approaches for WGG continue to be costly, primarily owing to the high expenses associated with library preparation and the laborious protocol. During prior development of foreground and background integrated genotyping by sequencing (FBI-seq), we discovered that any sequence-specific primer (SP) inherently possesses the capability to amplify a massive array of stable and reproducible non-specific PCR products across the genome. Here, we further improved FBI-seq by replacing the adapter ligated by Tn5 transposase with an arbitrary degenerate (AD) primer. The protocol for the enhanced FBI-seq unexpectedly mirrors a simplified thermal asymmetric interlaced (TAIL)-PCR, a technique that is widely used for isolation of flanking sequences. However, the improved TAIL-PCR maximizes the primer-template mismatched annealing capabilities of both SP and AD primers. In addition, leveraging of next-generation sequencing enhances the ability of this technique to assay tens of thousands of genome-wide loci for any species. This cost-effective, user-friendly, and powerful WGG tool, which we have named TAIL-PCR by sequencing (TAIL-peq), holds great potential for widespread application in breeding programs, thereby facilitating genome-assisted crop improvement.

Advancing broad bean true mosaic virus detection using conventional RT-PCR and real-time RT-PCR with novel primer set design.

Broad bean true mosaic virus (BBTMV) infects broad beans and peas, reducing yield. As BBTMV is transmitted through broad beans, many countries have implemented regulations to prevent the distribution of infected seeds. Currently, enzyme-linked immunosorbent assay (ELISA) is commonly used to detect BBTMV. While the PCR-based method is preferred for seed virus detection due to its sensitivity and speed. A BBTMV-specific PCR detection method has not yet been reported. A universal detection method currently exists that utilizes reverse transcription PCR (RT-PCR) for the Comovirus genus, to which BBTMV belongs. However, sequence analysis is required for species identification. To address this limitation, we developed and verified RT-PCR detection methods using newly designed BBTMV-specific primers. RT-PCR and real-time RT-PCR with these primers were approximately 5 × 105-106 times more sensitive than ELISA and 100-1000 times more sensitive than previously reported RT-PCR methods. Using RT-PCR and real-time RT-PCR employing these primers, we could detect BBTMV with same sensitivity when more than 3.0 × 105 copies were present per gram of broad bean seeds. Our newly developed detection methods can test for BBTMV with high sensitivity and speed.

Single primer site-specific nested PCR for accurate and rapid genome-walking.

Genome-walking is a molecular tool used to unveil uncharacterized DNA regions flanking a known DNA, which has been widely used in bioscience and related areas. This study developed a reliable and efficient PCR-based genome-walking approach, named as single primer site-specific nested PCR (SPN-PCR). A SPN-PCR set sequentially consists of three single-primer nested PCR amplifications. The primary relaxed thermal cycle promotes outmost nested site-specific primer (NSSP) to partially combine with numerous places on DNA template, synthesizing many single-stranded DNAs (ssDNA). Among them, the target ssDNA is exponentially amplified in the subsequent stringent cycles, as its 3' part possesses the outmost NSSP complement; but a non-target ssDNA cannot be amplified, because it does not possess such a complement. Stringent secondary and tertiary PCRs also exclusively enrich this target DNA. Finally, the target DNA product becomes predominant. The feasibility of SPN-PCR was validated by genome-walking several selected genes from two divergent species.

Rapid Detection of the Strawberry Foliar Nematode Aphelenchoides fragariae Using Recombinase Polymerase Amplification Assay with Lateral Flow Dipsticks.

Rapid and reliable diagnostic methods for plant-parasitic nematodes are critical for facilitating the selection of effective control measures. A diagnostic recombinase polymerase amplification (RPA) assay for Aphelenchoides fragariae using a TwistAmp® Basic Kit (TwistDx, Cambridge, UK) and AmplifyRP® Acceler8® Discovery Kit (Agdia, Elkhart, IN, USA) combined with lateral flow dipsticks (LF) has been developed. In this study, a LF-RPA assay was designed that targets the ITS rRNA gene of A. fragariae. This assay enables the specific detection of A. fragariae from crude nematode extracts without a DNA extraction step, and from DNA extracts of plant tissues infected with this nematode species. The LF-RPA assay showed reliable detection within 18-25 min with a sensitivity of 0.03 nematode per reaction tube for crude nematode extracts or 0.3 nematode per reaction tube using plant DNA extracts from 0.1 g of fresh leaves. The LF-RPA assay was developed and validated with a wide range of nematode and plant samples. Aphelenchoides fragariae was identified from seed samples in California. The LF-RPA assay has great potential for nematode diagnostics in the laboratory with minimal available equipment.

Multiplex PCR detection method of genetically modified canola event (MON94100, LBFLFK, and NS-B50027-4) combined with capillary electrophoresis.

Genetically modified organisms (GMOs) have been continuously developed for their convenience and productivity. In the past three years, three new GM canola events (MON94100, LBFLFK, and NS-B50027-4) have been developed. To efficiently control these GM canola events, the detection methods were needed. Therefore, the multiplex PCR method combined with capillary electrophoresis was developed for three GM canola events. Ten GM canola, eighteen GM soybean, thirty-two GM maize, and ten non-GM crops were used to evaluate the specificity of the method. The detection limit of the multiplex PCR assay was determined to be 0.005 ng in the DNA mixture and 0.1% in the spiked sample. The aim of this study was to establish multiplex PCR coupled with capillary electrophoresis for the newly produced three GM canola events. The developed method is expected to contribute to monitor the commercially available GM canola events. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01377-z.

A specific and rapid method for detecting Bacillus and Acinetobacter species in Daqu.

Daqu is a spontaneous, solid-state cereal fermentation product used for saccharification and as a starter culture for Chinese Baijiu production. Bacillus and Acinetobacter, two dominant microbial genera in Daqu, produce enzymes and organic acids that influence the Daqu quality. However, there are no rapid analytical methods for detecting Bacillus and Acinetobacter. We designed primers specific to the genera Bacillus and Acinetobacter to perform genetic comparisons using the 16 S rRNA. After amplification of polymerase chain reaction using specific primers, high-throughput sequencing was performed to detect strains of Bacillus and Acinetobacter. The results showed that the effective amplification rates for Bacillus and Acinetobacter in Daqu were 86.92% and 79.75%, respectively. Thus, we have devised and assessed a method to accurately identify the species associated with Bacillus and Acinetobacter in Daqu, which can also hold significance for bacterial typing and identification.

Argonaute-triggered visual and rebuilding-free foodborne pathogenic bacteria detection.

Foodborne pathogenic bacteria are recognized as the main causes of microbial contamination in food safety. Early screening and ultrasensitive detection of foodborne pathogenic bacteria is critical procedure to guarantee food safety. Argonaute is emerging as a new tool for detection owing to the programmability and high specificity. We reported a Novel and One-step cleavage method based on Argonaute by integrating Tag-specific primer extension and Exonuclease I (Exo I) for the first time, termed as NOTE-Ago. In this method, the invA of Salmonella typhi and nuc gene of Staphylococcus aureus were amplified using Tag-specific primer and the remaining primers were digested by Exo I. Then amplicons were served as the guide DNA for PfAgo. Consequently, the fluorophore-quencher reporter could be cleaved via PfAgo, resulting in changes in fluorescent intensity. With this strategy, target nucleic acid could be dexterously converted into fluorescent signals. The NOTE-Ago assay could detect 1 CFU/mL with a dynamic range from 1 to 108 CFU/mL. The satisfactory selectivity of NOTE-Ago assay further facilitated its application for detecting S. typhi- and S. aureus-contaminated food samples. This work enriches the toolbox of Argonaute-based detection and provides a one-step cleavage and rebuilding-free method for ultrasensitive detection of bacteria.

Prevalence of haemosporidia in Asian Glossy Starling with discovery of misbinding of Haemoproteus-specific primer to Plasmodium genera in Sarawak, Malaysian Borneo.

BACKGROUND: Plasmodium, Haemoproteus and Leucocytozoon are three mainly studied blood parasites known to cause malarial and pseudomalarial infections in avian worldwide. Although Sarawak is a biodiversity hotspot, molecular data on blood parasite diversity in birds are absent. The objective of the study is to determine the prevalence of blood parasite in Asian Glossy Starlings (AGS), an urban bird with high population density in Sarawak and to elucidate the phylogenetic relationship with other blood parasite. METHODS: Twenty-nine carcasses of juvenile AGS that were succumbed to death due to window collision were collected around the vicinity of Universiti Malaysia Sarawak. Nested-multiplex and nested PCR targeting the Cytochrome B gene were used to detect Plasmodium and Haemoproteus, and Leucocytozoon respectively. Two primer sets were used for Haemoproteus detection to increase detection sensitivity, with one being a genus-specific primer. RESULTS: Fourteen samples (prevalence rate: 48.28%) were found positive for avian Plasmodium. Phylogenetic analysis divided our sequences into five lineages, pFANTAIL01, pCOLL4, pACCBAD01, pALPSIS01 and pALPSIS02, with two lineages being novel. No Haemoproteus and Leucocytozoon was found in this study. However, Haemoproteus-specific primer used amplified our Plasmodium samples, making the primer non-specific to Haemoproteus only. CONCLUSION: This is the first blood parasite detection study on AGS using carcasses and blood clot as sample source in Sarawak. Due to the scarcity of longer sequences from regions with high genetic plasticity, usage of genus-specific primers should be validated with sequencing to ensure correct prevalence interpretation.

Stepwise Optimization of Real-Time RT-PCR Analysis.

Quantitative real-time reverse transcription PCR (qRT-PCR) analysis has been used routinely to quantify gene expression levels. Primer design and the optimization of qRT-PCR parameters are critical for the accuracy and reproducibility of qRT-PCR analysis. Computational tool-assisted primer design often overlooks the presence of homologous sequences of the gene of interest and the sequence similarities between homologous genes in a plant genome. This sometimes results in skipping the optimization of qRT-PCR parameters due to the false confidence in the quality of the designed primers. Here we present a stepwise optimization protocol for single nucleotide polymorphisms (SNPs)-based sequence-specific primer design and sequential optimization of primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference and target gene. The goal of this optimization protocol is to achieve a standard cDNA concentration curve with an R2 ≥ 0.9999 and efficiency (E) = 100 ± 5% for the best primer pair of each gene, which serves as the prerequisite for using the 2-ΔΔCT method for data analysis.

Expression and Localization of Fas-Associated Factor 1 in Testicular Tissues of Different Ages and Ovaries at Different Reproductive Cycle Phases of Bos grunniens.

Fas-associated factor 1 (FAF1), a member of the Fas family, is involved in biological processes such as apoptosis, inflammation, cell proliferation and proteostasis. This study aimed to explore the biological role of FAF1 in testicular tissue at different ages (juveniles (1 and 2 years old), adults (3, 4, 6, and 7 years old) and old-aged animals (11 years old)) and ovaries during different reproductive cycle phases (follicular, luteal, and pregnancy phases). FAF1 mRNA, relative protein expression and protein expression localization were determined in testes and ovaries using real-time quantification, WB and immunohistochemistry (IHC), respectively. Real-time quantification of testis tissues showed that the relative expression of FAF1 mRNA in testis tissues at 3, 4 and 7 years of age was significantly higher than of those in other ages, and in ovarian tissues was significantly higher in luteal phase ovaries than those in follicular and pregnancy phase ovaries; follicular phase ovaries were the lowest. WB of testis tissues showed that the relative protein expression of FAF1 protein was significantly higher at 11 and 7 years of age; in ovarian tissue, the relative protein expression of FAF1 protein was significantly higher in follicular phase ovaries than in luteal and pregnancy phase ovaries, and lowest in luteal phase ovaries. The relative protein expression of FAF1 at 3, 4 and 7 years of age was the lowest. IHC showed that FAF1 was mainly expressed in spermatozoa, spermatocytes, spermatogonia and supporting cells; in ovarian tissue, FAF1 was expressed in ovarian germ epithelial cells, granulosa cells, cumulus cells and luteal cells. The IHC results showed that FAF1 mRNA and protein were significantly differentially expressed in testes of different ages and ovarian tissues of different reproductive cycle phases, revealing the significance of FAF1 in the regulation of male and female B. grunniens reproductive physiology. Furthermore, our results provide a basis for the further exploration of FAF1 in the reproductive physiology of B. grunniens.

Semi-Site-Specific Primer PCR: A Simple but Reliable Genome-Walking Tool.

Genome-walking has been frequently applied to molecular biology and related areas. Herein, a simple but reliable genome-walking technique, termed semi-site-specific primer PCR (3SP-PCR), is presented. The key to 3SP-PCR is the use of a semi-site-specific primer in secondary PCR that partially overlaps its corresponding primary site-specific primer. A 3SP-PCR set comprises two rounds of nested amplification reactions. In each round of reaction, any primer is allowed to partially anneal to the DNA template once only in the single relaxed-stringency cycle, creating a pool of single-stranded DNAs. The target single-stranded DNA can be converted into a double-stranded molecule directed by the site-specific primer, and thus can be exponentially amplified by the subsequent high-stringency cycles. The non-target one cannot be converted into a double-strand due to the lack of a perfect binding site to any primer, and thus fails to be amplified. We validated the 3SP-PCR method by using it to probe the unknown DNA regions of rice hygromycin genes and Levilactobacillus brevis CD0817 glutamic acid decarboxylase genes.

Molecular diet analysis in mussels and other metazoan filter feeders and an assessment of their utility as natural eDNA samplers.

Molecular gut content analysis is a popular tool to study food web interactions and has recently been suggested as an alternative source for DNA-based biomonitoring. However, the overabundant consumer's DNA often outcompetes that of its diet during PCR. Lineage-specific primers are an efficient means to reduce consumer amplification while retaining broad specificity for dietary taxa. Here, we designed an amplicon sequencing assay to monitor the eukaryotic diet of mussels and other metazoan filter feeders and explore the utility of mussels as natural eDNA samplers to monitor planktonic communities. We designed several lineage-specific rDNA primers with broad taxonomic suitability for eukaryotes. The primers were tested using DNA extracts of different limnic and marine mussel species and the results compared to eDNA water samples collected next to the mussel colonies. In addition, we analysed several 25-year time series samples of mussels from German rivers. Our primer sets efficiently prevent the amplification of mussels and other metazoans. The recovered DNA reflects a broad dietary preference across the eukaryotic tree of life and considerable taxonomic overlap with filtered water samples. We also show the utility of a reversed version of our primers, which prevents amplification of nonmetazoan taxa from complex eukaryote community samples, by enriching fauna associated with the marine brown algae Fucus vesiculosus. Our protocol will enable large-scale dietary analysis in metazoan filter feeders, facilitate aquatic food web analysis and allow surveying of aquacultures for pathogens. Moreover, we show that mussels and other aquatic filter feeders can serve as complementary DNA source for biomonitoring.

Heat-Treated Meat Origin Tracing and Authenticity through a Practical Multiplex Polymerase Chain Reaction Approach.

Meat adulteration have become a global issue, which has increasingly raised concerns due to not only economic losses and religious issues, but also public safety and its negative effects on human health. Using optimal primers for seven target species, a multiplex PCR method was developed for the molecular authentication of camel, cattle, dog, pig, chicken, sheep and duck in one tube reaction. Species-specific amplification from the premixed total DNA of seven species was corroborated by DNA sequencing. The limit of detection (LOD) is as low as 0.025 ng DNA for the simultaneous identification of seven species in both raw and heat-processed meat or target meat: as little as 0.1% (w/w) of the total meat weight. This method is strongly reproducible even while exposed to intensively heat-processed meat and meat mixtures, which renders it able to trace meat origins in real-world foodstuffs based on the authenticity assessment of commercial meat samples. Therefore, this method is a powerful tool for the inspection of meat adulterants and has broad application prospects.

Allele Specific Polymerase Chain Reaction (AS-PCR) Assay to Detect Knockdown and Acetylcholinesterase Mutations in Anopheles superpictus

Objective: The study aims to determine the presence of L1014F, L1014S, L1014C alleles, which are responsible for knockdown resistance and Ace-1 G119S alleles, which are responsible for acetylcholinesterase insensitivity in Anopheles superpictus, the secondary vector of malaria in Turkey. Methods: In this study, 60 Anopheles superpictus adult females were collected from Aydin, Denizli, and Mugla provinces. Then, allele-specific primers for kdr L1014F, L1014S, and L1014C alleles, and the Ace-1 G119S allele were designed. The presence of these alleles was screened in three Anopheles superpictus populations by allele-specific polymerase chain reaction. Results: Although L1014S allele frequency was too low in Aydin, Mugla, and Denizli populations, neither kdr L1014F and L1014C nor Ace-1 G119S mutations were found in any population. Conclusion: In this study, kdr L1014S mutation was detected for the first time in the Aegean Anopheles superpictus populations.

Development of a Real-Time Quantitative PCR Assay for the Specific Detection of Bacillus velezensis and Its Application in the Study of Colonization Ability.

Bacillus velezensis is a widely used biocontrol agent closely related to B. amyloliquefaciens, and the two species cannot be distinguished by universal primers that are currently available. The study aimed to establish a rapid, specific detection approach for B. velezensis. Many unique gene sequences of B. velezensis were selected through whole genome sequence alignment of B. velezensis strains and were used to design a series of forward and reverse primers, which were then screened by PCR and qPCR using different Bacillus samples as templates. The colonization ability of B. velezensis ZF2 in different soils and different soil environmental conditions was measured by qPCR and a 10-fold dilution plating assay. A specific primer pair targeting the sequence of the D3N19_RS13500 gene of B. velezensis ZF2 was screened and could successfully distinguish B. velezensis from B. amyloliquefaciens. A rapid specific real-time qPCR detection system for B. velezensis was established. B. velezensis ZF2 had a very strong colonization ability in desert soil, and the optimal soil pH was 7-8. Moreover, the colonization ability of strain ZF2 was significantly enhanced when organic matter from different nitrogen sources was added to the substrate. This study will provide assistance for rapid specificity detection and biocontrol application of B. velezensis strains.