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PloS One 2024Alternative splicing (AS) is a universal phenomenon in eukaryotes, and it is still challenging to identify AS events. Several methods have been developed to identify AS...
Alternative splicing (AS) is a universal phenomenon in eukaryotes, and it is still challenging to identify AS events. Several methods have been developed to identify AS events, such as expressed sequence tags (EST), microarrays and RNA-seq. However, EST has limitations in identifying low-abundance genes, while microarray and RNA-seq are high-throughput technologies, and PCR-based technology is needed for validation. To overcome the limitations of EST and shortcomings of high-throughput technologies, we established a method to identify AS events, especially for low-abundance genes, by reverse transcription (RT) PCR with gene-specific primers (GSPs) followed by nested PCR. This process includes two major steps: 1) the use of GSPs to amplify as long as the specific gene segment and 2) multiple rounds of nested PCR to screen the AS and confirm the unknown splicing variants. With this method, we successfully identified three new splicing variants, namely, GenBank Accession No. HM623886 for the bdnf gene (GenBank GeneID: 12064), GenBank Accession No. JF417977 for the trkc gene (GenBank GeneID: 18213) and GenBank Accession No. HM623888 for the glb-18 gene (GenBank GeneID: 172485). In addition to its reliability and simplicity, the method is also cost-effective and labor-intensive. In conclusion, we developed an RT-nested PCR method using gene-specific primers to efficiently identify known and novel AS variants. This approach overcomes the limitations of existing methods for detecting rare transcripts. By enabling the discovery of new isoforms, especially for low-abundance genes, this technique can aid research into aberrant splicing in disease. Future studies can apply this method to uncover AS variants involved in cancer, neurodegeneration, and other splicing-related disorders.
Topics: Alternative Splicing; Humans; Brain-Derived Neurotrophic Factor; Reverse Transcriptase Polymerase Chain Reaction; DNA Primers
PubMed: 38935635
DOI: 10.1371/journal.pone.0305201 -
Microorganisms Jun 2024() is found in water, soil, plants and animals. Even though it has low virulence, it has increasingly been found to cause a number of infectious diseases in people with...
() is found in water, soil, plants and animals. Even though it has low virulence, it has increasingly been found to cause a number of infectious diseases in people with low immunity. The identification of mainly uses biochemical methods, such as the API 20NE or Vitek-2. The typing studies of have mainly utilized PFGE, rep-PCR, AFLP, 16s rDNA sequencing, RecA-PCR RFLP, and MALDI-TOF MS. This study aims to evaluate the polymorphisms of variable-number tandem-repeats (VNTRs) within genomic DNA of strains. The tandem repeats (TRs) in genomic DNA are discovered using Tandem Repeat Finder software (version 4.09). Twelve different VNTRs are designated and assigned to the nomenclature. The primers for PCR of 12 loci are designed. The PCR product size is converted to the number of tandem repeats in every locus. The relatedness of 65 strains from geographically different countries are analyzed by means of 12-variable-number tandem-repeat analysis(MLVA-12). A total of 51 different genotypes are found in 65 strains. These strains, which were collected from the same environmental samples, hospitals, and countries, are clustered within the same or closely genotypes. The MLVA-12 assay has a good discriminatory power for species determination, typing of , and inferring the origin of bacteria.
PubMed: 38930593
DOI: 10.3390/microorganisms12061211 -
Animals : An Open Access Journal From... Jun 2024is the primary cause of visceral and cutaneous leishmaniasis in the European Mediterranean region. Subspecies-level characterization of aids epidemiological studies by...
is the primary cause of visceral and cutaneous leishmaniasis in the European Mediterranean region. Subspecies-level characterization of aids epidemiological studies by offering insights into the evolution and geographical distribution of the parasite and reservoir identity. In this study, conducted in north-east Spain, 26 DNA samples of were analyzed, comprising 21 from 10 humans and 5 from 5 dogs. Minicircle kinetoplast DNA (kDNA) polymerase chain reaction assays using primers MC1 and MC2, followed by sequencing, were employed to assess intraspecific genetic variability. Single-nucleotide polymorphism (SNP) analysis detected seven genotypes (G1, G2, G12*-G15*, and G17*), with five being reported for the first time (*). The most prevalent was the newly described G13 (54%), while the other currently identified genotypes were predominantly found in single samples. The in silico restriction fragment length polymorphism (RFLP) method revealed five genotypes (B, F, N, P, and W), one of them previously unreported (W). Genotype B was the most prevalent (85%), comprising three SNP genotypes (G1, G2, and G13), whereas the other RFLP genotypes were associated with single SNP genotypes. These kDNA genotyping methods revealed significant intraspecific genetic diversity in , demonstrating their suitability for fingerprinting and strain monitoring.
PubMed: 38929415
DOI: 10.3390/ani14121796 -
Animals : An Open Access Journal From... Jun 2024Sex determination based just on morphological traits such as plumage dichromatism, sexual size dimorphism, behavior, or vocalizations is really challenging because of...
Sex determination based just on morphological traits such as plumage dichromatism, sexual size dimorphism, behavior, or vocalizations is really challenging because of the sexual monomorphism present in more than half of avian species. Currently, a lot of them can be tested through DNA-based procedures, but they do not fit all the avian species, such as . The aim of this study was to design a new molecular method suitable for routine sex determination for that species that is fast, simple, and cost- and time- effective. DNA was isolated from dry blood stain and/or chest feather samples of species. We used two sets of sex-specific primers (ZF/ZR and WF/WR) to amplify the expected fragments localized on the highly conserved gene to distinguish between sexes due to the W-specific DNA sequence present only in females. We confirmed the accuracy and consistency of the PCR-based method based on length differences to distinguish between the sexes of , which amplified two fragments in females and one fragment in males.
PubMed: 38929338
DOI: 10.3390/ani14121719 -
International Journal of Molecular... Jun 2024Polymerase Chain Reaction (PCR) amplification is widely used for retrieving information from DNA storage. During the PCR amplification process, nonspecific pairing...
Polymerase Chain Reaction (PCR) amplification is widely used for retrieving information from DNA storage. During the PCR amplification process, nonspecific pairing between the 3' end of the primer and the DNA sequence can cause cross-talk in the amplification reaction, leading to the generation of interfering sequences and reduced amplification accuracy. To address this issue, we propose an efficient coding algorithm for PCR amplification information retrieval (ECA-PCRAIR). This algorithm employs variable-length scanning and pruning optimization to construct a codebook that maximizes storage density while satisfying traditional biological constraints. Subsequently, a codeword search tree is constructed based on the primer library to optimize the codebook, and a variable-length interleaver is used for constraint detection and correction, thereby minimizing the likelihood of nonspecific pairing. Experimental results demonstrate that ECA-PCRAIR can reduce the probability of nonspecific pairing between the 3' end of the primer and the DNA sequence to 2-25%, enhancing the robustness of the DNA sequences. Additionally, ECA-PCRAIR achieves a storage density of 2.14-3.67 bits per nucleotide (bits/nt), significantly improving storage capacity.
Topics: Algorithms; Polymerase Chain Reaction; DNA; Information Storage and Retrieval; DNA Primers; Base Sequence
PubMed: 38928155
DOI: 10.3390/ijms25126449 -
Genes May 2024With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still...
With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-h), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-h on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future.
Topics: Erythropoietin; Animals; Mice; Doping in Sports; Dependovirus; Humans; Genetic Vectors; Male; Genetic Therapy; Models, Animal
PubMed: 38927645
DOI: 10.3390/genes15060709 -
Pathogens (Basel, Switzerland) May 2024has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children...
A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens.
has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR platform to detect sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of sp. were used. The limit of detection of the mini-PCR test was 1 fg/μL for DNA samples diluted in water, 10 fg/μL for /snail DNA scramble, and 100 fg/μL for /stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories.
PubMed: 38921738
DOI: 10.3390/pathogens13060440 -
Current Issues in Molecular Biology May 2024A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G...
A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G mutant-type plasmids were constructed, respectively. In this study, a PCR method based on the TaqMan amplification refractory mutation system was proposed to detect mtDNA 1555A>G. A common upstream primer, a common TaqMan probe, and two downstream allele-specific primers with mismatched bases were designed. One-step amplification and detection of the wild-type and mutant type at the 1555 site were realized for the deafness-related gene through two reactions. Based on this detection method, the minimum detection limit of the wild-type and mutant type detection systems for plasmids was 50 copies/μL. The minimum sensitivity for the detection of nucleic acids in real dried blood spot (DBS) samples was 0.1 ng/μL. In the normal DBS DNA sample, the detection limit of the mutation abundance reached 0.78%. The specificity of the detection method was 100%, and the coefficient of variation was less than 3.36%. This approach was validated using clinical DNA extracted from 113 DBS samples of newborns. Additionally, it showed 100% agreement with bi-directional Sanger sequencing. It can be used as an optional method for the clinical detection of deafness-related genes.
PubMed: 38920998
DOI: 10.3390/cimb46060326 -
Heliyon Jun 2024Periodontal disease is highly prevalent in both humans and dogs. Although there have been reports of cross-infection of periodontopathic bacteria, methods for assessing...
Periodontal disease is highly prevalent in both humans and dogs. Although there have been reports of cross-infection of periodontopathic bacteria, methods for assessing it have yet to be established. The actual status of cross-infection remains to be seen. The purpose of this study was to evaluate the utility of bacterial DNA and serum immunoglobulin G (IgG) antibody titer assays to assess infection of human-pathogenic and dog-pathogenic species in dogs. Four experimental beagles were used for establishing methods. Sixty-six companion dogs at veterinary clinics visiting for treatment and prophylaxis of periodontal disease were used and divided into healthy, gingivitis, and periodontitis groups. Periodontal pathogens such as and were investigated as target bacteria. DNA levels of both bacteria were measured using species-specific primers designed for real-time polymerase chain reaction (PCR). Serum IgG titers of both bacteria were measured by enzyme-linked immunosorbent assay (ELISA). PCR primers were confirmed to have high sensitivity and specificity. However, there was no relationship between the amount of bacterial DNA and the severity of the periodontal disease. In addition, dogs with periodontitis had higher IgG titers against both bacteria compared to dogs in the healthy and gingivitis groups; there was cross-reactivity between the two bacteria. Receiver operating characteristic (ROC) analysis of IgG titers against both bacteria showed high sensitivity (>90 %) and specificity (>75 %). Since both bacteria were distinguished by DNA assays, the combination of these assays may be useful in the evaluation of cross-infection.
PubMed: 38919974
DOI: 10.1016/j.heliyon.2024.e31872 -
Scientific Reports Jun 2024Sugarcane smut is the most damaging disease that is present almost across the globe, causing mild to severe yield losses depending upon the cultivar types, pathogen...
Sugarcane smut is the most damaging disease that is present almost across the globe, causing mild to severe yield losses depending upon the cultivar types, pathogen races and climatic conditions. Cultivation of smut-resistant cultivars is the most feasible and economical option to mitigate its damages. Previous investigations revealed that there is a scarcity of information on early detection and effective strategies to suppress etiological agents of smut disease due to the characteristics overlapping within species complexes. In this study, 104 sugarcane cultivars were screened by artificial inoculation with homogenate of all possible pathogen races of Sporisorium scitamineum during two consecutive growing seasons. The logistic smut growth pattern and the disease intrinsic rate were recorded by disease growth curve. Variable levels of disease incidence i.e., ranging from 0 to 54.10% were observed among these sugarcane cultivars. Besides, pathogen DNA in plant shoots of all the cultivars was successfully amplified by PCR method using smut-specific primers except 26 cultivars which showed an immune reaction in the field trial. Furthermore, the plant germination and tillering of susceptible sugarcane cultivars were greatly influenced by pathogen inoculation. In susceptible cultivars, S. scitamineum caused a significant reduction in setts germination, coupled with profuse tillering, resulting in fewer millable canes. Correlation analysis demonstrated that there was a positive relationship between reduction in setts germination and increase in the number of tillers. The present study would be helpful for the evaluation of smut resistance in a wide range of sugarcane germplasm, especially from the aspects of setts germination and tillers formation, and it also screened out several excellent germplasm for potential application in sugarcane breeding.
Topics: Saccharum; Plant Diseases; Germination; Disease Resistance; Ustilaginales
PubMed: 38918529
DOI: 10.1038/s41598-024-64810-1