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Chinese rice‐field eels rhabdovirus (CrERV) causes haemorrhagic disease in Chinese rice‐field eels (Monopterus albus), leading to significant mortality and economic losses. Sensitive detection of CrERV nucleic acids is essential to control the spread of this pathogen and to treat infected individuals. Herein, we developed an efficient and sensitive droplet digital PCR (ddPCR) method to rapidly detect and quantify CrERV. The ddPCR assay optimal conditions were an annealing temperature of 53°C, and primer and probe concentrations of 0.5 and 0.25 μM, respectively. The assay had a diagnostic sensitivity of 0.23 copies/μL, and was highly specific, showing no cross reactivity with other viruses (infectious haematopoietic necrosis virus, grass carp reovirus, spring viremia of carp virus, largemouth bass ranavirus, carp edema virus, Chinese giant salamander iridovirus, and white spot syndrome virus). Real‐time quantitative PCR testing of 30 Chinese rice‐field eels samples detected CrERV in 7 samples (23.3%), whereas ddPCR detected CrERV in 12 samples (40%), demonstrating its higher sensitivity. Thus, ddPCR represents an advanced method to absolutely quantify CrERV in infected fish with low virus concentrations, providing a valuable tool to manage the spread and impact of CrERV. [ABSTRACT FROM AUTHOR]

Our study concentrated on the expression of miRNA-375 in the hypothalamic-pituitary-gonadal axis of female Hu sheep. The investigation involved cloning the precursor sequence of miR-NA-375, followed by comparison with database entries and subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In our approach, we obtained ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus, and pineal gland from fertile but nonpregnant Hu ewes. MiRNA extraction kit was used to extract miRNA from the above eight tissues. Real-time fluorescent quantitative polymerase chain reaction was used to evaluate the role of miR-375 in the hy-pothalamic-pituitary-gonadal axis. The results of miR-375 precursor sequence cloning were compared with those of Anopheles gambiae, Apis mellifera, Bos taurus, Drosophila melanogaster, Danio rerio, Fugu rubripes, Gallus gallus, Homo sapiens, Monodelphis domestica, Macaca mulatta, Mus musculus, Pan troglodytes, Rattus norvegicus, Tetraodon nigroviridis, Xenopus tropicalis miR-375 in miRBase database. It was found that oar-miR-375 was highly conserved. Notably, miR-375 expression in the pineal gland was significantly higher (p < 0.01) than that in the ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus. The study also involved predicting miR-375 target genes. GO and KEGG enrichment analyses of these predicted target genes revealed that miR-375 is involved in 182 biological processes, affects 186 cellular components, and participates in 184 molecular functions. In terms of pathway enrichment, miR-375 was linked to nine pathways, including the Hippo, Wnt, and mTOR signaling pathways. This study has validated the interaction between miR-375 and its target gene FZD4, which can be recognized and bound to produce effects. These findings lead to the inference that miR-375 may play a crucial regulatory role in sheep reproduction through the Hippo pathway and Wnt pathway, laying a foundation for further exploration of miR-375's role in this domain. [ABSTRACT FROM AUTHOR]

Auricularia heimuer is a wood-rotting edible mushroom, and with the continuous development of the industry, the research on its grass-rotting cultivation is becoming more and more important. In this study, A. heimuer was cultivated using herbaceous substrate (reed) completely replacing the traditional woody substrate (oak), and the correlation between the relative expression of cellulase gene, cellulase activity, cellulose degradation and yield of different strains of A. heimuer were studied by combining qRT-PCR technology at different growth stages. The results showed that the cellulose degradation were positively correlated with the yield of reed and sawdust substrate at two growth stages, and were positively correlated with three cellulase activities. The relative expression of four cellulase genes were positively correlated with enzyme activity. There were inter-strain differences in the expression of the enzyme genes, which were basically consistent with the trend of the enzyme activity of the strains; g5372 and g7270 were more actively expressed in the mycelium period, while g9664 and g10234 were more actively expressed in the fruiting period. The results of SEM showed that the mycelium of A15 and A125 were different in their ability to degrade and utilize lignocellulose in reed substrate. The parental hybridization test further verified that qRT-PCR could be used as a rapid method to evaluate the cellulose degradation ability of A. heimuer strains. Seven strains (A12, A15, A184, A224, Z6, Z12, and Z18) with high cellulose degradation ability were screened. This study provides a reference for further understanding the role of A. heimuer cellulase genes in the degradation and metabolism of cellulose and for breeding new varieties more suitable for herbaceous substrate cultivation. [ABSTRACT FROM AUTHOR]

Abstract Our study concentrated on the expression of miRNA-375 in the hypothalamic-pituitary-gonadal axis of female Hu sheep. The investigation involved cloning the precursor sequence of miR-NA-375, followed by comparison with database entries and subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In our approach, we obtained ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus, and pineal gland from fertile but nonpregnant Hu ewes. MiRNA extraction kit was used to extract miRNA from the above eight tissues. Real-time fluorescent quantitative polymerase chain reaction was used to evaluate the role of miR-375 in the hy-pothalamic-pituitary-gonadal axis. The results of miR-375 precursor sequence cloning were compared with those of Anopheles gambiae, Apis mellifera, Bos taurus, Drosophila melanogaster, Danio rerio, Fugu rubripes, Gallus gallus, Homo sapiens, Monodelphis domestica, Macaca mulatta, Mus musculus, Pan troglodytes, Rattus norvegicus, Tetraodon nigroviridis, Xenopus tropicalis miR-375 in miRBase database. It was found that oar-miR-375 was highly conserved. Notably, miR-375 expression in the pineal gland was significantly higher (p

Abstract Reverse transcription-quantitative PCR (RT-qPCR) is widely used for monitoring viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in wastewater. Various materials, including plasmid DNA, synthetic nucleic acids, PCR amplicons, genomic DNA, and cDNA, are currently used for SARS-CoV-2 quantification by generating standard curves. We assessed three common standards on quantifying SARS-CoV-2 RNA across nine wastewater treatment plants in Finland, as part of the national wastewater surveillance effort. We pairwise compared RT-qPCR results from 148 wastewater samples, using both IDT (#10006625, IDT, USA) and CODEX standards (#SC2-RNAC-1100, CODEX DNA), and 179 samples using both IDT and EURM019 standards (#EURM-019, European Commission, Joint Research Centre) in our assessment. Amongst the tested standards, the CODEX standard consistently yielded more stable results than either the IDT or EURM019 standards. We found that SARS-CoV-2 levels were higher with the IDT standard (4.36 Log10 GC/100 mL) compared to the CODEX standard (4.05 Log10 GC/100 mL). Similarly, quantification using the IDT standard was higher (5.27 Log10 GC/100 mL) than values obtained with the EURM019 (4.81 Log10 GC/100 mL). SARS-CoV-2 RNA quantified with IDT and CODEX standards exhibited stronger concordance (Spearman’s correlation rho median of 0.79) compared to those quantified with IDT and EURM019 standards (rho median of 0.59). This study highlights the significant impact of standard material selection on SARS-CoV-2 RNA quantification, emphasizing the need for harmonization in standard material.

Paper Mulberry (Broussonetia papyrifera) possesses medicinal, economic, and ecological significance and is extensively used for feed production, papermaking, and ecological restoration due to its ease of propagation, rapid growth rate, and strong stress resistance. The recent completion of the sequencing of the Paper Mulberry genome has prompted further research into the genetic breeding and molecular biology of this important species. A highly stable reference gene is essential to enhance the quantitative analysis of functional genes in Paper Mulberry; however, none has been identified. Accordingly, in this study, the leaves, stems, roots, petioles, young fruits, and mature fruits of Paper Mulberry plants were selected as experimental materials, and nine candidate reference genes, namely, α-TUB1, α-TUB2, β-TUB, H2A, ACT, DnaJ, UBQ, CDC2, and TIP41, were identified by RT-qPCR. Their stability was assessed using the geNorm, Normfinder, Delta Ct, BestKeeper, and RefFinder algorithms, identifying ACT and UBQ as showing the greatest stability. The expression of BpMYB090, which regulates the production of trichomes, was examined in the leaves of plants of the wild type (which have more trichomes) and mutant (which have fewer trichomes) at various developmental stages to validate the results of this study. As a result, their identification addresses a critical gap in the field of Paper Mulberry research, providing a solid foundation for future research that will concentrate on the characterization of pertinent functional genes in this economically valuable species.

【Objective】Real-time quantitative PCR is one of the most conventional and efficient techniques for studying the mRNA expression level of target genes. The proper selection of reference genes is of great significance for the accuracy of the results. The study aimed to identify the most suitable reference genes by evaluating the expression stability of candidate reference genes in different samples of Spodoptera frugiperda larvae under azadirachtin stress. The results provided a basis for functional analysis of target genes and molecular toxicological mechanism analysis of azadirachtin.【Method】Eight candidate reference genes, including α-TUB, β-1-TUB, Actin, EF1α, EF2, GAPDH, RPL3 and RPL3, of S. frugiperda were selected. The expression stability of these genes in different samples of larvae, cuticle, midgut, fat body and malpighian tube under azadirachtin stress was evaluated by the ΔCt, GeNorm, NormFinder, BestKeeper and RefFinder software. And the optimal number of reference genes were determined by GeNorm software. Finally, the best reference gene combinations in different samples were comfirmed.【Result】When exposed to azadirachtin stress, in larvae samples, the expression stability of α-TUB and β-1-TUB was the highest by ΔCt and Normfinder analyses; the expression stability of RPL3 and RPL13 was the highest by GeNorm analysis; and the expression stability of α-TUB and RPL13 was the highest by BestKeeper analysis. In cuticle samples, β-1-TUB and α-TUB were rated as the most stable genes in ΔCt, GeNorm and Normfinder, while Actin and β-1-TUB were the most stable in BestKeeper. In the midgut tissue, ΔCt analysis found that EF2 and β-1-TUB expression was the most stable; GeNorm assessment showed that EF1α and RPL3 expression was the most stable; Normfinder considered that EF2 and β-1-TUB expression was the most stable, and BestKeeper analysis found that RPL13 and RPL3 expression was the most stable. ΔCt, GeNorm and Normfinder analyses showed that RPL3 and EF1α were the most stable in fat body tissue, while BestKeeper analysis showed that RPL13 and RPL3 were the most stable. In malpighian tube tissue, ΔCt analysis found that RPL3 and EF2 expression was the most stable, GeNorm assessment indicated that EF1α and EF2 expression was the most stable; Normfinder considered that β-1-TUB and EF2 expression was the most stable, and BestKeeper analysis found that RPL13 and β-1-TUB expression was the most stable. In addition, GeNorm software analysis found that the optimal number of reference genes in each sample was 2. Therefore, as assessed by RefFinder comprehensive analysis, α-TUB and β-1-TUB had the highest expression stability in larvae and cuticle tissue samples; EF2 and RPL3 had the highest expression stability in larval midgut and malpighian tube samples; and RPL3 and EF1α had the highest expression stability in fat body samples.【Conclusion】Under azadirachtin stress, the optimal reference genes for different samples of S. frugiperda larvae were shown as: α-TUB and β-1-TUB for larvae and cuticle samples; EF2 and RPL3 for midgut and malpighian tube samples; and RPL3 and EF1α for fat body samples.

Liquidambar formosana Hance is renowned for its rich leaf color and possesses notable advantages, such as robust adaptability, strong resistance to diseases and pests, and rapid growth, making it a preferred choice for urban greening and carbon sequestration forest initiatives. The completion of whole-genome sequencing of L. formosana has spurred an increased interest in exploring the molecular mechanisms underlying seasonal changes in leaf color, marking a significant focus in L. formosana breeding research. However, there is currently a lack of stable reference genes suitable for analyzing the expression patterns of functional genes in L. formosana exhibiting varying leaf colors. This study selected five L. formosana varieties with significant differences in leaf colors. Through the RT-qPCR analysis, and evaluation using BestKeeper, geNorm, NormFinder, Delta Ct, and RefFinder, the expression stability of 14 candidate reference genes was examined. Consequently, two reference genes (LifEF1-α and LifACT) with stable expression, suitable for RT-qPCR of L. formosana with diverse leaf colors, were identified. The stability of these selected reference genes was further validated by examining the LifbHLH137 gene, which promoted the biosynthesis of anthocyanins. This advancement facilitated molecular biology and genetic breeding investigations of L. formosana, providing essential data for the precise quantification of functional genes associated with leaf color variation.

As one of the developed genetically modified (GM) maize varieties in China, CC-2 has demonstrated promising commercial prospects during demonstration planting. The establishment of detection methods is a technical prerequisite for effective supervision and regulation of CC-2 maize. In this study, we have developed an event-specific quantification method that targets the junction region between the exogenous gene and the 5' flanking genomic DNA (gDNA) of CC-2. The accuracy and precision of this method were evaluated across high, medium, and low levels of CC-2 maize content, revealing biases within ±25% and satisfactory precision data. Additionally, we determined the limits of quantification of the method to be 0.05% (equivalent to 20 copies) of the CC-2 maize. A collaborative trial further confirmed that our event-specific method for detecting CC-2 produces reliable, comparable, and reproducible results when applied to five different samples provided by various sources. Furthermore, we calculated the expanded uncertainty associated with determining the content level of CC-2 in these samples. [ABSTRACT FROM AUTHOR]

【Objective】Real-time quantitative PCR is one of the most conventional and efficient techniques for studying the mRNA expression level of target genes. The proper selection of reference genes is of great significance for the accuracy of the results. The study aimed to identify the most suitable reference genes by evaluating the expression stability of candidate reference genes in different samples of Spodoptera frugiperda larvae under azadirachtin stress. The results provided a basis for functional analysis of target genes and molecular toxicological mechanism analysis of azadirachtin.【Method】 Eight candidate reference genes, including α-TUB, β-1-TUB, Actin, EF1α, EF2, GAPDH, RPL3 and RPL3, of S. frugiperda were selected. The expression stability of these genes in different samples of larvae, cuticle, midgut, fat body and malpighian tube under azadirachtin stress was evaluated by the ΔCt, GeNorm, NormFinder, BestKeeper and RefFinder software. And the optimal number of reference genes were determined by GeNorm software. Finally, the best reference gene combinations in different samples were comfirmed.【Result】When exposed to azadirachtin stress, in larvae samples, the expression stability of α-TUB and β-1-TUB was the highest by ΔCt and Normfinder analyses; the expression stability of RPL3 and RPL13 was the highest by GeNorm analysis; and the expression stability of α-TUB and RPL13 was the highest by BestKeeper analysis. In cuticle samples, β-1-TUB and α-TUB were rated as the most stable genes in ΔCt, GeNorm and Normfinder, while Actin and β-1-TUB were the most stable in BestKeeper. In the midgut tissue, ΔCt analysis found that EF2 and β-1-TUB expression was the most stable; GeNorm assessment showed that EF1α and RPL3 expression was the most stable; Normfinder considered that EF2 and β-1- TUB expression was the most stable, and BestKeeper analysis found that RPL13 and RPL3 expression was the most stable. ΔCt, GeNorm and Normfinder analyses showed that RPL3 and EF1α were the most stable in fat body tissue, while BestKeeper analysis showed that RPL13 and RPL3 were the most stable. In malpighian tube tissue, ΔCt analysis found that RPL3 and EF2 expression was the most stable, GeNorm assessment indicated that EF1α and EF2 expression was the most stable; Normfinder considered that β-1-TUB and EF2 expression was the most stable, and BestKeeper analysis found that RPL13 and β-1- TUB expression was the most stable. In addition, GeNorm software analysis found that the optimal number of reference genes in each sample was 2. Therefore, as assessed by RefFinder comprehensive analysis, α-TUB and β-1-TUB had the highest expression stability in larvae and cuticle tissue samples; EF2 and RPL3 had the highest expression stability in larval midgut and malpighian tube samples; and RPL3 and EF1α had the highest expression stability in fat body samples.【Conclusion】Under azadirachtin stress, the optimal reference genes for different samples of S. frugiperda larvae were shown as: α-TUB and β-1-TUB for larvae and cuticle samples; EF2 and RPL3 for midgut and malpighian tube samples; and RPL3 and EF1α for fat body samples. [ABSTRACT FROM AUTHOR]

Simple Summary: Gene expression analysis by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) can provide strong evidence for scientists to understand the molecular mechanisms underlying various physiological processes. Selecting appropriate reference genes under specific experimental conditions are prerequisites for achieving the accurate results of qRT-PCR. Here, the expression stability of seven reference genes was evaluated in Chilo sacchariphagus, a destructive pest of sugarcane, under different experimental conditions, encompassing tissues, temperatures, and sexes. The expression patterns of C. sacchariphagus pheromone binding protein 1 gene (CsacPBP1) across three different experimental conditions mentioned above were evaluated to verify the results. The findings of this study will lay an important foundation for the future study on functional gene expressions in the pest. Chilo sacchariphagus (Lepidoptera: Pyralidae) is an economically important sugarcane pest. Although numerous studies were conducted on the physiological responses in C. sacchariphagus, little is known regarding the genes regulating these physiological processes. Gene expression analysis by qRT-PCR can offer a significant indication for functional gene studies. To our knowledge, the reference genes of C. sacchariphagus have not been screened or evaluated, which hinders the functional gene study. In the present study, the stability of seven reference genes (β-ACT, GAPDH, BTF3, 28S, RPL7, EF1α, and SDHA) was evaluated in C. sacchariphagus under different experimental conditions, including tissues (antenna, head, thorax, abdomen, leg, and wing), temperatures (4 °C, 25 °C, and 37 °C) and sexes (male and female), through RefFinder, which integrates four algorithms (Normfinder, BestKeeper, ΔCt method, and geNorm). The findings suggested that the combination of β-ACT and RPL7 is ideal to analyze gene expressions in different tissues and at distinct temperatures, and EF1α and SDHA were suitable reference genes for comparing gene expressions between sexes. Finally, the expression profiles of CsacPBP1 gene were evaluated, and the outcomes further confirm the importance of selecting fitting reference genes for normalization of qRT-PCR data. This study represents the first kind in screening out suitable reference genes for gene expression analysis in C. sacchariphagus. Information from this study is poised to galvanize future inquiry into the gene expression of C. sacchariphagus, an economically important pest of sugarcane. [ABSTRACT FROM AUTHOR]

Objective: To investigate the relationship between the DNA methylation state of NRG1 promoter and its expression changes, and to analyze the clinical significance of its regulatory mechanism of DNA methylation in cervical carcinoma. Methods: This was a retrospective study. One-hundred and twenty patients from the Department of Gynecology of Cangzhou People's Hospital from September 2017 to September 2019 were selected, including 40 cases of cervical SCC, 40 cases of high grade squamous intraepithelial lesions (HSIL) and 40 cases of control cervical tissues. RT-qPCR, immunohistochemistry and DNA methylation-specific PCR (MSP) were used to detect the mRNA and protein expression of NRG1 and DNA methylation status in different tissue types. Results: Immunohistochemical results showed that the positive protein expression rate of NRG1 gene in the SCC group was lower than that in both HSIL and Control groups. RT-qPCR results showed that the mRNA gene of NRG1 gradually decreased in expression with the increase of cervical tissue lesions, with a statistically significant difference. Similarly, it also found that the mRNA expression level of NRG1 in the SCC group was independent of patients' age (p>0.05), but significantly correlated with tumor pathological staging, surgical pathology staging and lymphatic metastasis (p<0.05). Furthermore, methylation-specific PCR results revealed a significantly higher DNA methylation rate of NRG1 gene in the SCC group than in both HSIL and Control groups, with a statistically significant difference. Moreover, the methylation degree of NRG1 gene in SCC tissues was negatively correlated with its mRNA expression (p<0.05). Conclusions: Abnormal DNA hypermethylation of NRG1 gene inhibits the expression of mRNA and protein in the progression of cervical tissue from normal to cancerous state, which is involved in the occurrence and development of cervical carcinoma. [ABSTRACT FROM AUTHOR]

The stem tips of Zizania latifolia at different development stages were used as research materials. The expression stability of nine candidate reference genes (ACT1, H2B, UBI, EF-1α, GAPDH, β-actin, 60S, SKIP and AQP) were evaluated using qRT-PCR. The data were analyzed with GeNorm and NormFinder software. Present results indicated that the expression of ACT1 was stable and that it could be used as the optimal reference gene for studying the development stage of gall formation. ACT1 was selected as the reference gene to verify the expression level of the correlative genes in the gall formation stage of Z. latifolia. Our results were consistent with the previous transcriptome sequencing results. This study revealed that ACT1 was the classic reference gene for the analysis of correlative genes in all of the gall development stages of Z. latifolia. [ABSTRACT FROM AUTHOR]

The thermotolerant yeast Pichia kudriavzevii (previously known as Issatchenkia orientalis), can produce ethanol from a variety of carbon sources and grows at around 45 °C. Thus, this yeast is considered a useful biocatalyst for producing ethanol from lignocellulose through simultaneous saccharification and fermentation (SSF). SSF has several advantages, such as a simplified manufacturing process, ease of operation and reduced energy input. Using P. kudriavzevii NBRC1279 and NBRC1664, we previously succeeded in producing ethanol through SSF; however, the extent to which inhibitors by-produced from lignocellulose hydrolysis affect the growth and ethanol productivity of the two strains remains to be investigated. In this study, to better understand the inhibitor tolerance capacity of the two strains, spot assay, growth experiment, real-time quantitative PCR (RT-qPCR) analysis and multiple sequence alignment analysis were carried out. When P. kudriavzevii NBRC1279 and NBRC1664, as well as Saccharomyces cerevisiae BY4742 as a control, were cultured on SCD plates containing 17% ethanol, 42 mM furfural, 56 mM 5-hydroxymethylfurfural (HMF) or 10 mM vanillin, only P. kudriavzevii NBRC1664 was able to grow under all conditions. Moreover, the inhibitor tolerance capacity of P. kudriavzevii NBRC1664 was greater than those of other strains using SCD medium containing the same concentrations of various inhibitors. When an RT-qPCR analysis of seven gene sequences from aldehyde dehydrogenase and the aldehyde dehydrogenase family protein (ADHF) was performed using P. kudriavzevii NBRC1664 cultivated in the presence of 56 mM HMF, ADHF1 and ADHF2 were up-regulated in the early logarithmic growth phase. Moreover, a multiple sequence alignment of the amino acid sequences of ADHF1, ADHF2 and the known ADH suggested that ADHF1 and ADHF2 may catalyze the reversible NAD+-dependent oxidation of HMF. Our data may be useful for future studies on the metabolic engineering of more useful strains for ethanol production from lignocellulose. [ABSTRACT FROM AUTHOR]

Background: The involvement of N7-Methylguanine (m7G) RNA methylation regulators in the progression of different types of solid cancers in humans has been established. However, the specific impact of m7G-related genes on Acute myeloid leukemia (AML) remains uncertain. Our research aims to build a novel signature of M7Gs that could enhance our understanding of the molecular heterogeneity in leukemia.Methods: The RNA-seq and clinical data of patients with AML were acquired from the UCSC XENA website. Prognostic-related genes were selected using LASSO to construct a risk-scoring model. External datasets were utilized to validate the effectiveness of the model, and the mRNA expressions of candidate genes were measured using RT-qPCR.Results: A prognostic model was developed using a risk-scoring approach based on three candidate genes (IFIT5, EIF4E2, and LARP1) and their respective risk coefficients. Multivariate Cox regression analysis revealed a significant association between the risk score and overall survival (p

Determination of the genomic copy number of Cre transgene is critical for the establishment of transgenic animal models. We demonstrate a real-time quantitative polymerase chain reaction (RT-qPCR) based method for genomic copy number determination of improved Cre (iCre), which was tested in a transgenic mouse model of CYP19A1-iCre. The standards were derived from tandem repeats of iCre fragments which were constructed by a new application of megaprimer PCR. Two rounds of megaprimer PCR were used to obtain specific tandem repeats of iCre fragments, which were subsequently cloned into pUCm-T vectors. After the fragments of IL-2 (reference gene) were ligated into the same vectors, standards with copy number ratios (CNR) of 0.5, 1, 2, 4, 8, and 16 between iCre and IL-2 were completed. The R2 values of the standard curves exceeded 0.99. The calculated CNR value of the quality control sample was satisfactory. Multiple copies of iCre were detected in founder mice. After several passages, the copy number of iCre gradually decreased to a single copy, which was consistent with theoretical expectation. The established method is a universal and sensitive program for genomic copy number determination of iCre in transgenic mice.

Taking type A H1N1 inactivated influenza virus as an example, a rapid detection method with high sensitivity, specificity, biosafety and easy industrialization was established, referred as colorimetric assay using magnetic crosslinking precipitation coupled with gold nanoparticles（MCPGNP）. Firstly, the efficient capture of the inactivated virus was achieved by performing the chemical coupling between the activated carboxyl group on the magnetic bead and the primary amine group of the protein on the surface of the inactivated virus. Secondly, the aptamer was incubated with magnetic beads containing the captured viruses, followed by the magnetic separation and heat elution of the beadbound aptamer. The aptamer specifically recognized the H1N1 inactivated virus. Thirdly, the polymerase chain reaction （PCR） was performed to amplify the eluted aptamer. Finally, the colorimetric detection of the H1N1 inactivated virus was realized, which was based on the different binding affinity of single-strand primers and double-strand amplified products on GNP. When H5N1 inactivated virus was used as the blank control, the detection of limit（S/N=3）was 1 ng/L, which was 1/900 of the detection limit of the specific electrochemical impedance sensor previously reported by our research group. The colorimetric detection of clinical H1N1 inactivated virus positive throat swab samples with low viral load was also achieved for the first time. Under non-optimized conditions, the full process from virus capture to detection was completed within 3 h, demonstrating the clinical application value of MCP-GNP. In addition, all the results of colorimetric detection in this study were consistent with those of fluorescence quantitative PCR, which confirmed the reliability of our method. The core innovation of this work is the use of chemical coupling reaction to conveniently achieve the capture of inactivated virus at ultra-low concentration without the need of viral nucleic acid extraction and enrichment. In addition, the DNA aptamer targeting the H1N1 inactivated virus, instead of the active virus, was used for the specific recognition of the virus, enabling the high biosafety. [ABSTRACT FROM AUTHOR]

Phaseolus vulgaris L. , commonly known as the common bean, is a highly nutritious crop often called the "poor man's meat". However, it is susceptible to various diseases throughout the cropping season, with anthracnose caused by Colletotrichum lindemuthianum being a significant threat that leads to substantial losses. There is still a lack of understanding about the molecular basis of C. lindemuthianum pathogenicity. The first step in understanding this is to identify pathogenicity genes that express more during infection of common beans. A reverse transcription quantitative real-time PCR (qPCR) method can be used for virulence gene expression. However, this approach requires selecting appropriate reference genes to normalize relative gene expression data. Currently, there is no reference gene available for C. lindemuthianum. In this study, we selected eight candidate reference genes from the available genome of C. lindemuthianum to bridge the gap. These genes were ACT (Actin), β-tub (β-tubulin), EF (Elongation Factor), Cyt C (Cytochrome C), His H3 (Histone H3), CHS1 (Chitin synthetase), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and abfA (Alpha- l -Arabinofuranosidase A). The primers for these candidate reference genes were able to amplify cDNA only from the pathogen, demonstrating their specificity. The qPCR efficiency of the primers ranged from 80% to 103%. We analyzed the stability of gene expression in C. lindemuthianum by exposing the mycelium to nine different stress conditions. We employed algorithms, such as GeNorm, NormFinder, BestKeeper, and RefFinder tools, to identify the most stable gene. The analysis using these tools revealed that EF , GAPDH , and β-tub most stable genes, while ACT and CHS1 showed relatively low expression stability. A large number of potential effector genes have been identified through bioinformatics analysis in C. lindemuthianum. The stable genes for qPCR (EF and GAPDH) discovered in this study will aid the scientific community in determining the relative expression of C. lindemuthianum effector genes. • Gene expression investigations are hampered by the lack of reference genes in C. lindemuthianum. • We provide the list of qPCR genes that can be used to perform expression analysis of genes in C. lindemuthianum. • We identified EF and GAPDH as the most stable genes. • Our results will aid the scientific community in determining the relative expression of C. lindemuthianum effector genes. [ABSTRACT FROM AUTHOR]

Killer cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) play crucial roles in regulating NK cell activity. Here, we report a real-time quantitative PCR (qPCR) to genotype all KIR genes and their copy numbers simultaneously. With 18 pairs of locus-specific primers, we identified KIR genes by Ct values and determined KIR copy number using the 2−∆Ct method. Haplotypes were assigned based on KIR gene copy numbers. The real-time qPCR results were consistent with the NGS method, except for one sample with KIR2DL5 discrepancy. qPCR is a multiplex method that can identify KIR copy number, which helps obtain a relatively accurate haplotype structure, facilitating increased KIR research in laboratories where NGS or other high-resolution methods are not available. [ABSTRACT FROM AUTHOR]

Auricularia heimuer is a wood-rotting edible mushroom, and with the continuous development of the industry, the research on its grass-rotting cultivation is becoming more and more important. In this study, A. heimuer was cultivated using herbaceous substrate (reed) completely replacing the traditional woody substrate (oak), and the correlation between the relative expression of cellulase gene, cellulase activity, cellulose degradation and yield of different strains of A. heimuer were studied by combining qRT-PCR technology at different growth stages. The results showed that the cellulose degradation were positively correlated with the yield of reed and sawdust substrate at two growth stages, and were positively correlated with three cellulase activities. The relative expression of four cellulase genes were positively correlated with enzyme activity. There were inter-strain differences in the expression of the enzyme genes, which were basically consistent with the trend of the enzyme activity of the strains; g5372 and g7270 were more actively expressed in the mycelium period, while g9664 and g10234 were more actively expressed in the fruiting period. The results of SEM showed that the mycelium of A15 and A125 were different in their ability to degrade and utilize lignocellulose in reed substrate. The parental hybridization test further verified that qRT-PCR could be used as a rapid method to evaluate the cellulose degradation ability of A. heimuer strains. Seven strains (A12, A15, A184, A224, Z6, Z12, and Z18) with high cellulose degradation ability were screened. This study provides a reference for further understanding the role of A. heimuer cellulase genes in the degradation and metabolism of cellulose and for breeding new varieties more suitable for herbaceous substrate cultivation.

Determination of the genomic copy number of Cre transgene is critical for the establishment of transgenic animal models. We demonstrate a real-time quantitative polymerase chain reaction (RT-qPCR) based method for genomic copy number determination of improved Cre (iCre), which was tested in a transgenic mouse model of CYP19A1-iCre. The standards were derived from tandem repeats of iCre fragments which were constructed by a new application of megaprimer PCR. Two rounds of megaprimer PCR were used to obtain specific tandem repeats of iCre fragments, which were subsequently cloned into pUCm-T vectors. After the fragments of IL-2 (reference gene) were ligated into the same vectors, standards with copy number ratios (CNR) of 0.5, 1, 2, 4, 8, and 16 between iCre and IL-2 were completed. The R2 values of the standard curves exceeded 0.99. The calculated CNR value of the quality control sample was satisfactory. Multiple copies of iCre were detected in founder mice. After several passages, the copy number of iCre gradually decreased to a single copy, which was consistent with theoretical expectation. The established method is a universal and sensitive program for genomic copy number determination of iCre in transgenic mice. [ABSTRACT FROM AUTHOR]

In traditional Chinese medicine, Angelica dahurica is a valuable herb with numerous therapeutic applications for a range of ailments. There have not yet been any articles on the methodical assessment and choice of the best reference genes for A. dahurica gene expression studies. Real-time quantitative PCR (RT-qPCR) is widely employed as the predominant method for investigating gene expression. In order to ensure the precise determination of target gene expression outcomes in RT-qPCR analysis, it is imperative to employ stable reference genes. In this study, a total of 11 candidate reference genes including SAND family protein (SAND), polypyrimidine tract-binding protein (PTBP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), TIP41-like protein (TIP41), cyclophilin 2 (CYP2), elongation factor 1 α (EF1α), ubiquitin-protein ligase 9 (UBC9), tubulin β-6 (TUB6), thioredoxin-like protein YLS8 (YLS8), and tubulin-α (TUBA) were selected from the transcriptome of A. dahurica. Subsequently, three statistical algorithms (geNorm, NormFinder, and BestKeeper) were employed to assess the stability of their expression patterns across seven distinct stimulus treatments. The outcomes obtained from these analyses were subsequently amalgamated into a comprehensive ranking using RefFinder. Additionally, one target gene, phenylalanine ammonia-lyase (PAL), was used to confirm the effectiveness of the selected reference genes. According to the findings of this study, the two most stable reference genes for normalizing the expression of genes in A. dahurica are TIP41 and UBC9. Overall, our research has determined the appropriate reference genes for RT-qPCR in A. dahurica and provides a crucial foundation for gene screening and identifying genes associated with the biosynthesis of active ingredients in A. dahurica. [ABSTRACT FROM AUTHOR]

Minimal residual disease (MRD) has become an essential tool in the management of B-cell acute lymphoblastic leukemia (B-ALL) and aids in tailoring treatment strategies to suit specific patient needs. Although much progress has been made in this area, there is limited data on the use of MRD in the Indian context. Our objective was to identify relevant literature that discusses the utility of MRD in the management of B-cell ALL in adolescents and young adults (AYA) and adults in Indian settings. A systematic search and screening of articles were performed using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The primary data source was PubMed followed by Google Scholar for articles and conference proceedings. Of the 254 records screened, 24 records were retained for analysis. MRD monitoring had a significant role in the management of AYA/adult B-cell ALL patients. Variability of results was observed across these studies with respect to methods, techniques, and use. However, these studies evidenced and validated the importance of MRD assessment in risk-adapted management of B-cell ALL and highlighted the need for optimization. The advances in MRD diagnostics and applications are yet to be tested and adopted in Indian settings. Hence, there is a need for in-depth research to develop and optimize approaches for calibrating country-specific management strategies. The potential role of MRD assessments in anticipating relapse or treatment failures warrants more attention for the preemptive positioning of novel strategies involving immunotherapies. [ABSTRACT FROM AUTHOR]

【Objective】Capsicum chinense is one of the main cultivated species of pepper, which generally has a strong fruit aroma formed by branched chain esters. AAT (alcohol acyl-CoA transferase) catalyzes the last step in the synthesis of branched chain esters and has an important impact on its content. Identification of the AAT gene family in C. chinense and analysis on its expression patterns will provide references for its functional studies.【Method】Bioinformatics and real-time quantitative PCR analysis were applied to identify the AAT gene family of C. chinense and analyze its expression patterns.【Result】Ten members of the AAT gene family were identified and named CcAAT1-CcAAT10 according to their distribution order on six chromosomes. Physicochemical properties prediction revealed that the length of amino acid sequence encoded by AAT gene family range from 256 to 683 aa, with their molecular mass ranging from 29 to 77 kDa, and isoelectric point ranging from 5.34 to 8.79. Average hydrophobic coefficients of them were all negative values. Their instability indexes ranged 23.76 to 51.02. Protein structure prediction showed that their secondary structure was dominated by α-helices and irregular convolutions, and the tertiary structure varied widely. Subcellular localization prediction revealed that all CcAAT were located in the cytoplasm and 16 cis-regulatory elements were found on their gene promoter. Spatiotemporal expression analysis showed that CcAAT5 and CcAAT6 had no detectable expression, CcAAT8 was specifically expressed in flowers and fruits, CcAAT1, CcAAT3 and CcAAT7 were highly expressed in leaves, and CcAAT4 was highly expressed in roots.【Conclusion】It clarified the expression pattern of the AAT gene family in C. chinense and it was hypothesized that CcAAT8 might be a key gene affecting the content of branched chain esters.

The transcription factor activating protein 2α (AP2α) is a nuclear transcription factor that specifically binds to DNA and is involved in the regulation of animal embryonic development, cell growth, apoptosis, tumorigenesis, immunity, and other biological processes. In our previous study, the transcription factor AP2α was discovered as a key disease-resistance candidate gene in the yellow drum, Nibea albiflora, in response to a Vibrio harveyi infection through genome-wide association analysis. In the present study, the AP2α gene, which encodes a protein of 424 amino acids, was cloned from a yellow drum. The N-terminal is a trans-activation domain rich in proline and glutamine (P/Q-rich domain), and the middle is a central basic structure (central basic region), which is a highly conserved helix-span-helix motif responsible for DNA binding and protein dimerization. Multiple alignments of amino acid sequences showed that AP2α was highly conserved, with a homology of more than 84.63% among the detected fish, amphibians, birds, and mammals. Quantitative RT-PCR demonstrated that AP2α was widely distributed in the nine tested tissues, with the highest expression in the blood. Moreover, its transcription was significantly activated in the liver, spleen, and head kidney by V. harveyi infection, especially in the liver wherein the transcript level of AP2α reached a peak at 24 h post infection. Subcellular localization by constructing the recombinant eukaryotic expression plasmid pGEFP-AP2α and transfection into HEK293T cells revealed that AP2α was localized in the nucleus. In addition, the soluble GST-AP2α fusion protein was expressed in Escherichia coli BL21. These results demonstrate that AP2α plays an important role in the immune response against V. harveyi in N. albiflora. We provide new insights into the role of AP2α in the innate immunity of teleost fishes and provide a basis for studies on immune mechanisms and disease-resistant breeding in N. albiflora and other marine fish.

The study evaluated expression profiles of few regulatory lncRNAs in oral squamous cell carcinoma and normal mucosa adjacent to oral cancer using paired fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissues stored at a different duration of time (1–5 years) using real-time quantitative PCR. The quantity and quality of total RNA isolated from FFPE tissues was less compared with that of fresh frozen tissues, which resulted in a noncorrelation of quantification cycle values. Following normalization, the expression of lncRNAs in the paired tissues did not differ significantly. The differential expression of the lncRNAs in the study was consistent with The Cancer Genome Atlas head and neck squamous cell carcinoma database. The study findings demonstrate the possibility of performing accurate quantitative analysis of lncRNAs using short amplicons and standardized real-time quantitative PCR assays in oral squamous cell carcinoma FFPE samples.

Chilo sacchariphagus (Lepidoptera: Pyralidae) is an economically important sugarcane pest. Although numerous studies were conducted on the physiological responses in C. sacchariphagus, little is known regarding the genes regulating these physiological processes. Gene expression analysis by qRT-PCR can offer a significant indication for functional gene studies. To our knowledge, the reference genes of C. sacchariphagus have not been screened or evaluated, which hinders the functional gene study. In the present study, the stability of seven reference genes (β-ACT, GAPDH, BTF3, 28S, RPL7, EF1α, and SDHA) was evaluated in C. sacchariphagus under different experimental conditions, including tissues (antenna, head, thorax, abdomen, leg, and wing), temperatures (4 °C, 25 °C, and 37 °C) and sexes (male and female), through RefFinder, which integrates four algorithms (Normfinder, BestKeeper, ΔCt method, and geNorm). The findings suggested that the combination of β-ACT and RPL7 is ideal to analyze gene expressions in different tissues and at distinct temperatures, and EF1α and SDHA were suitable reference genes for comparing gene expressions between sexes. Finally, the expression profiles of CsacPBP1 gene were evaluated, and the outcomes further confirm the importance of selecting fitting reference genes for normalization of qRT-PCR data. This study represents the first kind in screening out suitable reference genes for gene expression analysis in C. sacchariphagus. Information from this study is poised to galvanize future inquiry into the gene expression of C. sacchariphagus, an economically important pest of sugarcane.

The stem tips of Zizania latifolia at different development stages were used as research materials. The expression stability of nine candidate reference genes (ACT1, H2B, UBI, EF-1α, GAPDH, β-actin, 60S, SKIP and AQP) were evaluated using qRT-PCR. The data were analyzed with GeNorm and NormFinder software. Present results indicated that the expression of ACT1 was stable and that it could be used as the optimal reference gene for studying the development stage of gall formation. ACT1 was selected as the reference gene to verify the expression level of the correlative genes in the gall formation stage of Z. latifolia. Our results were consistent with the previous transcriptome sequencing results. This study revealed that ACT1 was the classic reference gene for the analysis of correlative genes in all of the gall development stages of Z. latifolia.

Abstract Background Porcine circovirus 4 (PCV4), a newly emerging virus that was first discovered in 2019, may pose a potential threat to the pig industry. Droplet digital PCR (ddPCR) is an absolute quantitative method that has high sensitivity and accuracy. In this study, we developed a novel ddPCR assay to detect PCV4. Furthermore, we evaluated the detection limit, sensitivity, specificity and reproducibility of the ddPCR and TaqMan real-time quantitative PCR (qPCR) and tested 160 clinical samples to compare the detection rate of the two methods. Results The detection limit for ddPCR was 0.54 copies/µL, 10.6 times greater sensitivity than qPCR. Both ddPCR and qPCR assays exhibited good linearity and repeatability, and the established ddPCR method was highly specific for PCV4. The results of clinical sample testing showed that the positivity rate of ddPCR (5.6%) was higher than that of qPCR (4.4%). Conclusions This study successfully developed a sensitive, specific and repeatable ddPCR assay for PCV4 detection, which can be widely used in clinical diagnosis of PCV4 infections.

The global prevalence of inflammatory bowel disease (IBD) is on the rise, prompting significant attention from researchers worldwide. IBD entails chronic inflammatory disorders of the intestinal tract, characterized by alternating flares and remissions. Through high-throughput sequencing, numerous studies have unveiled a potential microbial signature for IBD patients showing intestinal enrichment of oralassociated bacteria. Simultaneously, the oral microbiome can be perturbed by intestinal inflammation. Our prior investigation, based on 16S rRNA amplicon sequencing, underscored elevated abundance of Veillonella spp. and Prevotella spp. in the salivary microbiomes of IBD patients. Noteworthy, Prevotella salivae emerged as a distinct species significantly associated with IBD. P. salivae is an under-recognized pathogen that was found to play a role in both oral and systemic diseases. In this study, we delve deeper into the salivary microbiomes of both IBD patients and healthy controls. Employing diverse cultivation techniques and realtime quantitative polymerase chain reactions (RT-qPCR), we gauged the prevalence and abundance of Veillonella spp., Prevotella spp., and P. salivae. Our isolation efforts yielded 407 and 168 strains of Veillonella spp., as well as 173 and 90 strains of Prevotella spp., from the saliva samples of IBD patients and healthy controls, respectively. Veillonella-vancomycin agar emerged as the discerning choice for optimal Veillonella spp. cultivation, while Schaedler kanamycin-vancomycin agar proved to be the most suitable medium for cultivating Prevotella spp. strains. Comparing our RT-qPCR findings to the previous 16S rRNA amplicon sequencing data, the results corroborated the higher abundance of Veillonella spp., Prevotella spp., and P. salivae in the saliva of IBD patients compared to healthy controls. However, it's worth noting that in contrast to RT-qPCR, the 16S rRNA amplicon sequencing data revealed greater absolute abundance of all three bacterial groups in both IBD patients and controls. [ABSTRACT FROM AUTHOR]

Aphidoletes aphidimyza is a predator that is an important biological agent used to control agricultural and forestry aphids. Although many studies have investigated its biological and ecological characteristics, few molecular studies have been reported. The current study was performed to identify suitable reference genes to facilitate future gene expression and function analyses via quantitative reverse transcription PCR. Eight reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RPS13, RPL8, RPS3, α-Tub, β-actin, RPL32, and elongation factor 1 alpha (EF1-α) were selected. Their expression levels were determined under four different experimental conditions (developmental stages, adult tissues, sugar treatment, and starvation treatment) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper, and RefFinder). The results showed that GAPDH, RPL32, and EF1-α were ranked as the best reference gene combinations for measuring gene expression levels among different developing stages and in various starvation treatments. RPL8 and RPS3 were recommended to normalize the gene expression levels among different adult tissues. RPL32, β-actin, and EF1-α were recommended sugar-feeding conditions. To validate the utility of the selected reference pair, RPL8, and RPS3, we estimated the tissue-biased expression level of a chemosensory protein gene (AaphCSP1). As expected, AaphCSP1 is highly expressed in the antennae and lowly expressed in the abdomen. These findings will lay the foundation for future research on the molecular physiology and biochemistry of A. aphidimyza. [ABSTRACT FROM AUTHOR]

Real-time quantitative PCR (qPCR) has been widely used to quantify gene copy numbers in microbial ecology. Despite its simplicity and straightforwardness, establishing qPCR assays is often impeded by the tedious process of producing qPCR standards by cloning the target DNA into plasmids. Here, we designed double-stranded synthetic DNA fragments from consensus sequences as qPCR standards by aligning microbial gene sequences (10-20 sequences per gene). Efficiency of standards from synthetic DNA was compared with plasmid standards by qPCR assays for different phylogenetic marker and functional genes involved in carbon (C) and nitrogen (N) cycling, tested with DNA extracted from a broad range of soils. Results showed that qPCR standard curves using synthetic DNA performed equally well to those from plasmids for all the genes tested. Furthermore, gene copy numbers from DNA extracted from soils obtained by using synthetic standards or plasmid standards were comparable. Our approach therefore demonstrates that a synthetic DNA fragment as qPCR standard provides comparable sensitivity and reliability to a traditional plasmid standard, while being more time- and cost-efficient. [ABSTRACT FROM AUTHOR]

【Objective】 Capsicum chinense is one of the main cultivated species of pepper, which generally has a strong fruit aroma formed by branched chain esters. AAT (alcohol acyl-CoA transferase) catalyzes the last step in the synthesis of branched chain esters and has an important impact on its content. Identification of the AAT gene family in C. chinense and analysis on its expression patterns will provide references for its functional studies.【Method】Bioinformatics and real-time quantitative PCR analysis were applied to identify the AAT gene family of C. chinense and analyze its expression patterns.【Result】Ten members of the AAT gene family were identified and named CcAAT1-CcAAT10 according to their distribution order on six chromosomes. Physicochemical properties prediction revealed that the length of amino acid sequence encoded by AAT gene family range from 256 to 683 aa, with their molecular mass ranging from 29 to 77 kDa, and isoelectric point ranging from 5.34 to 8.79. Average hydrophobic coefficients of them were all negative values. Their instability indexes ranged 23.76 to 51.02. Protein structure prediction showed that their secondary structure was dominated by α-helices and irregular convolutions, and the tertiary structure varied widely. Subcellular localization prediction revealed that all CcAAT were located in the cytoplasm and 16 cis-regulatory elements were found on their gene promoter. Spatiotemporal expression analysis showed that CcAAT5 and CcAAT6 had no detectable expression, CcAAT8 was specifically expressed in flowers and fruits, CcAAT1, CcAAT3 and CcAAT7 were highly expressed in leaves, and CcAAT4 was highly expressed in roots.【Conclusion】It clarified the expression pattern of the AAT gene family in C. chinense and it was hypothesized that CcAAT8 might be a key gene affecting the content of branched chain esters. [ABSTRACT FROM AUTHOR]

Most hops are produced in Europe; therefore, it is alarming that the citrus bark cracking viroid (CBCVd), the causal agent of the severe hop stunt disease, was detected in different nonadjacent hop growing countries. It is still unclear how the initial infection occurred since CBCVd is typically found in citrus and not in hops. To extent data for a viroid risk assessment, potential hosts were tested for the presence of viroids in grocery stores in the hop producing areas of Slovenia and Germany. Samples positive for hop-pathogenic viroids were further used for infection studies. The surveys covered CBCVd, hop stunt viroid (HSVd), citrus exocortis viroid (CEVd), citrus dwarfing viroid (CDVd), citrus viroid V (CVdV), and citrus bent leaf viroid (CBLVd). The results show that all tested viroids can be found in fruits sold in grocery stores, thus there is a risk of introducing CBCVd, HSVd, and other viroids into the hop growing regions via imported fruits and their remains. Furthermore, the transmission study reveals that CBCVd and HSVd infected citrus fruits can lead to infected plants, irrespective of the type of inoculum whether in the form of RNA extract, injected sap, or fruit peel in the soil. Finally, the phylogenetic analysis showed that the sequence diversity within viroid samples is high and that CBCVd and HSVd sequence variants can be found, which are almost identical to variants confirmed in hop. We assumed that fruit imports contribute to international viroid spreading and inappropriate handling like fruit waste deposition to agricultural lands is a serious risk factor. [ABSTRACT FROM AUTHOR]

Equine piroplasmosis (EP) is a parasitic disease caused by Theileria equi (T. equi), Babesia caballi (B. caballi) and Theileria haneyi (T. haneyi). This disease is considered to be reportable by the World Organization for Animal Health (WOAH). Real-time quantitative PCR (qPCR) is regarded as a straightforward, rapid and sensitive diagnostic method to detect pathogens. However, qPCR has not been employed in the various epidemiological investigations of T. haneyi. In this study, we developed a new qPCR method to detect T. haneyi based on the chr1sco (chromosome 1 single-copy open reading frame (ORF)) gene, which has no detectable orthologs in T. equi or B. caballi. A TaqMan MGB probe was used in the development of the qPCR assay. A plasmid containing the chr1sco gene was constructed and used to establish the standard curves. The novel qPCR technique demonstrated great specificity for detecting additional frequent equine infectious pathogens and sensitivity for detecting diluted standard plasmids. This qPCR was further validated by comparison with an optimized nested PCR (nPCR) assay in the analysis of 96 clinical samples. The agreement between the nPCR assay and the established qPCR assay was 85.42%. The newly established method could contribute to the accurate diagnosis of T. haneyi infections in horses. [ABSTRACT FROM AUTHOR]

Real-time quantitative PCR (RT-qPCR) has a high sensitivity and strong specificity, and is widely used in the analysis of gene expression. Selecting appropriate internal reference genes is the key to accurately analyzing the expression changes of target genes by RT-qPCR. To find out the most suitable internal reference genes for studying the gene expression in Broussonetia papyrifera under abiotic stresses (including drought, salt, and ZnSO4 treatments), seven different tissues of B. papyrifera, as well as the roots, stems, and leaves of B. papyrifera under the abiotic stresses were used as test materials, and 15 candidate internal reference genes were screened based on the transcriptome data via RT-qPCR. Then, the expression stability of the candidate genes was comprehensively evaluated through the software geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), and RefFinder. The best internal reference genes and their combinations were screened out according to the analysis results. rRNA and Actin were the best reference genes under drought stress. Under salt stress, DOUB, HSP, NADH, and rRNA were the most stable reference genes. Under heavy metal stress, HSP and NADH were the most suitable reference genes. EIF3 and Actin were the most suitable internal reference genes in the different tissues of B. papyrifera. In addition, HSP, rRNA, NADH, and UBC were the most suitable internal reference genes for the abiotic stresses and the different tissues of B. papyrifera. The expression patterns of DREB and POD were analyzed by using the selected stable and unstable reference genes. This further verified the reliability of the screened internal reference genes. This study lays the foundation for the functional analysis and regulatory mechanism research of genes in B. papyrifera. [ABSTRACT FROM AUTHOR]

Capripox viruses (CaPVs), including sheep pox virus (SPV), goat pox virus (GPV), and lumpy skin disease virus (LSDV), are the cause of sheep pox (SPP), goat pox (GTP), and lumpy skin disease (LSD) in cattle. These diseases are of great economic significance to farmers, as they are endemic on farms and are a major constraint to international trade in livestock and their products. Capripoxvirus (CaPV) infections produce similar symptoms in sheep and goats, and the three viruses cannot be distinguished serologically. In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) method for identifying CaPV in goats, sheep, and cattle. Clinical samples were tested and verified. The developed assay was highly specific for target viruses, including GPVSPV and LSDV, which had no cross-reaction with other viruses causing similar clinical symptoms. An artificially synthesized positive control plasmid using the CaPV 32 gene inserted into the vector pMD19-T was used as a template, and the correlation coefficient of the linear regression curve (R2) was 0.9916, the estimated amplification efficiency (E) was 96.06%, and the sensitivity (limit of detection, LOD) was 3.80 copies per reaction. Using the clinical samples as a template, the limit of detection (LOD) was 4.91 × 10−5 ng per reaction (1.60 × 10−5–2.13 × 10−3 ng, 95% confidence interval (CI)), which means that this method was one of the most sensitive detection assays for CaPVs. A total of 85 clinical samples from CaPV-infected animals (goats, sheep, and cattle) and 50 clinical samples from healthy animals were used to test and compare the diagnostic results using the Synergy Brands (SYBR) Green-based PCR method recommended by the World Organization of Animal Health (WOAH). Both diagnostic sensitivity (DSe) (95.8–100%, 95% CI) and diagnostic specificity (DSp) (92.9–100%, 95% CI) results of the real-time quantitative PCR (qPCR) and SYBR Green PCR were 100%, and the kappa value (κ) was 1.0 (1-1, 95% CI). In summary, the assay established based on TaqMan probes was advantageous in high specificity, sensitivity, and general applicability and could be a competitive candidate tool for the diagnosis of CaPV in clinically suspected animals. [ABSTRACT FROM AUTHOR]

The thermotolerant yeast Pichia kudriavzevii (previously known as Issatchenkia orientalis), can produce ethanol from a variety of carbon sources and grows at around 45 °C. Thus, this yeast is considered a useful biocatalyst for producing ethanol from lignocellulose through simultaneous saccharification and fermentation (SSF). SSF has several advantages, such as a simplified manufacturing process, ease of operation and reduced energy input. Using P. kudriavzevii NBRC1279 and NBRC1664, we previously succeeded in producing ethanol through SSF; however, the extent to which inhibitors by-produced from lignocellulose hydrolysis affect the growth and ethanol productivity of the two strains remains to be investigated. In this study, to better understand the inhibitor tolerance capacity of the two strains, spot assay, growth experiment, real-time quantitative PCR (RT-qPCR) analysis and multiple sequence alignment analysis were carried out. When P. kudriavzevii NBRC1279 and NBRC1664, as well as Saccharomyces cerevisiae BY4742 as a control, were cultured on SCD plates containing 17% ethanol, 42 mM furfural, 56 mM 5-hydroxymethylfurfural (HMF) or 10 mM vanillin, only P. kudriavzevii NBRC1664 was able to grow under all conditions. Moreover, the inhibitor tolerance capacity of P. kudriavzevii NBRC1664 was greater than those of other strains using SCD medium containing the same concentrations of various inhibitors. When an RT-qPCR analysis of seven gene sequences from aldehyde dehydrogenase and the aldehyde dehydrogenase family protein (ADHF) was performed using P. kudriavzevii NBRC1664 cultivated in the presence of 56 mM HMF, ADHF1 and ADHF2 were up-regulated in the early logarithmic growth phase. Moreover, a multiple sequence alignment of the amino acid sequences of ADHF1, ADHF2 and the known ADH suggested that ADHF1 and ADHF2 may catalyze the reversible NAD+-dependent oxidation of HMF. Our data may be useful for future studies on the metabolic engineering of more useful strains for ethanol production from lignocellulose.

ABSTRACT Equine infectious anemia (EIA) has a worldwide distribution and causes severe economic losses to the equine industry. The EIA virus (EIAV) genome sequences from different countries are highly diverse, which poses a great challenge for pathogen identification with PCR. Phylogenetic analysis showed that although gag is the most conserved structural gene, it still has great genome variability. Currently, most existing PCR methods are designed based on the gag gene sequence and therefore do not cover all the viral strains, especially Asian EIAV strains. In this study, we developed a tat-gag-based real-time quantitative PCR (TG-qPCR) for the detection of EIAV by targeting the fragment between the tat and gag genes, which was relatively conserved in all the known EIAV strains. The performance of the TG-qPCR was evaluated against that of the standard qPCR (recommended by WOAH) by testing viral RNA extracted from viral supernatants of EIAVDLV2-6 and EIAVUK3, proviral DNA from peripheral blood mononuclear cells of artificially immunized horses, and virus nucleic acid from EIAV positive serum samples. The TG-qPCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the TG-qPCR assay was 1 copy/reaction for both viral RNA and proviral DNA based on the Poisson distribution. Compared to the qPCR, the TG-qPCR has better inclusivity and can detect not only Asian EIAV strains but also almost all the representative EIAV strains from other continents. The above results show that the TG-qPCR assay could serve as an effective tool for the early diagnosis of clinical EIA disease. IMPORTANCE Equine infectious anemia (EIA) has a worldwide distribution and causes significant losses to the equine industry worldwide. A reliable detection method is necessary to control the transmission of EIA virus (EIAV). Currently, most of the available real-time PCR assays, including the qPCR of recommended by WOAH, are developed according to the sequences of European or American EIAV strains; however, the primers and probe sequences have low homology with Asian EIAV strains. To the best of our knowledge, no qPCR method capable of the well detection of Asian EIAV strains, especially Chinese EIAV strains, has been published to date. The development of a sensitive, specific, and rapid qPCR assay for the detection of the EIAV strains is therefore of great importance.

The global prevalence of inflammatory bowel disease (IBD) is on the rise, prompting significant attention from researchers worldwide. IBD entails chronic inflammatory disorders of the intestinal tract, characterized by alternating flares and remissions. Through high-throughput sequencing, numerous studies have unveiled a potential microbial signature for IBD patients showing intestinal enrichment of oral-associated bacteria. Simultaneously, the oral microbiome can be perturbed by intestinal inflammation. Our prior investigation, based on 16S rRNA amplicon sequencing, underscored elevated abundance of Veillonella spp. and Prevotella spp. in the salivary microbiomes of IBD patients. Noteworthy, Prevotella salivae emerged as a distinct species significantly associated with IBD. P. salivae is an under-recognized pathogen that was found to play a role in both oral and systemic diseases. In this study, we delve deeper into the salivary microbiomes of both IBD patients and healthy controls. Employing diverse cultivation techniques and real-time quantitative polymerase chain reactions (RT-qPCR), we gauged the prevalence and abundance of Veillonella spp., Prevotella spp., and P. salivae. Our isolation efforts yielded 407 and 168 strains of Veillonella spp., as well as 173 and 90 strains of Prevotella spp., from the saliva samples of IBD patients and healthy controls, respectively. Veillonella-vancomycin agar emerged as the discerning choice for optimal Veillonella spp. cultivation, while Schaedler kanamycin-vancomycin agar proved to be the most suitable medium for cultivating Prevotella spp. strains. Comparing our RT-qPCR findings to the previous 16S rRNA amplicon sequencing data, the results corroborated the higher abundance of Veillonella spp., Prevotella spp., and P. salivae in the saliva of IBD patients compared to healthy controls. However, it’s worth noting that in contrast to RT-qPCR, the 16S rRNA amplicon sequencing data revealed greater absolute abundance of all three bacterial groups in both IBD patients and controls.

Reed canary grass (Phalaris arundinacea L.) is known for its tolerance to drought, heavy metals, and waterlogging, making it a popular choice for forage production and wetland restoration in the Qinghai-Tibet Plateau (QTP). To accurately assess gene expression in reed canary grass under different abiotic stresses, suitable reference genes need to be identified and validated. Thirteen candidate reference gene sequences were selected and screened using RT-qPCR to detect their expression levels in reed canary grass leaves under drought, salt, cadmium, and waterlogging stresses. Four algorithms were used to assess the stability of the expression levels of the candidate reference genes. The most stably expressed genes were UBC and H3 under drought Cd, ETF and CYT under salt stress, and ETF and TUB under waterlogging stress. GAPDH was found to be less stable under abiotic stresses. PIP-1, PAL, NAC 90, and WRKY 72A were selected as response genes for quantitative expression assessment under drought, salt, Cd, and waterlogging stresses to confirm the accuracy of the selected stable reference genes. These results provide a theoretical reference for assessing gene expression in reed canary grass under abiotic stresses. [ABSTRACT FROM AUTHOR]

Background: Porcine circovirus 4 (PCV4), a newly emerging virus that was first discovered in 2019, may pose a potential threat to the pig industry. Droplet digital PCR (ddPCR) is an absolute quantitative method that has high sensitivity and accuracy. In this study, we developed a novel ddPCR assay to detect PCV4. Furthermore, we evaluated the detection limit, sensitivity, specificity and reproducibility of the ddPCR and TaqMan real-time quantitative PCR (qPCR) and tested 160 clinical samples to compare the detection rate of the two methods. Results: The detection limit for ddPCR was 0.54 copies/µL, 10.6 times greater sensitivity than qPCR. Both ddPCR and qPCR assays exhibited good linearity and repeatability, and the established ddPCR method was highly specific for PCV4. The results of clinical sample testing showed that the positivity rate of ddPCR (5.6%) was higher than that of qPCR (4.4%). Conclusions: This study successfully developed a sensitive, specific and repeatable ddPCR assay for PCV4 detection, which can be widely used in clinical diagnosis of PCV4 infections. [ABSTRACT FROM AUTHOR]

Anaerobic/anoxia sequencing batch reactor (A/ASBR) system was used to analyze the quantity and composition of each branch of phosphorus accumulating organisms (PAOs) in activated sludge under different nitrate-nitrogen (NO−3-N) concentrations by using real-time quantitative polymerase chain reaction (PCR) technology. The study determined whether NO−3-N and its concentration change were the main driving factors for the variation of the quantity and composition of each branch of PAOs. The results show that with the increase of NO−3-N concentration from 10 mg/L to 40 mg/L, the number of bacterial 16S rRNA genes in the A/ASBR reactor changed slightly at 6.81×1011–7.53×1011 copies/g dry sludge. The number of PAO genes (Acc 16S rRNA) increased from 1.98×1011 to 3.53×1011 copies/g dry sludge, and the total number of ppk1 genes increased from 1.25×1011 to 3.59×1011 copies/g dry sludge. Additionally, the number of polyphosphate kinase (ppk) genes in Accumulibacter branch IA, IIC and IID was high, and the changes were positively related to the concentration of NO−3-N, while the number of branches in IIA, IIB and IIF was very low. The dosing concentration of NO−3-N was the main driving factor for the change of PAOs and their branch number and composition in the A/ASBR reactor. [ABSTRACT FROM AUTHOR]

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Abstract Background The HTLV-1 prevalence in China varies geographically, while HTLV-2 infection has rarely been found so far. Proviral load is one of the determining factors of pathogenesis and progression of HTLV-1 related diseases. However, neither molecular assays nor commercial kits are available for HTLV-1 diagnosis in China. The objective of the present study was to develop and validate a TaqMan qPCR assay for HTLV-1 proviral load quantification. Results A plasmid containing both the HTLV-1 of interest and a fragment of the RNase P (RPPH1) gene was constructed and used to establish the standard curves. The assay has a wide dynamic range (2.5 × 108 copies/reaction ~ 25 copies/reaction) and sensitive to 1 copy for HTLV-1 and RPPH1. The limit of detection for Hut102 cell concentration was 0.0218% (95% confidence interval 0.0179–0.0298%). The assay gave coefficient of variation (CV) for both the HTLV-1 and RPPH1 Ct values. All of the HTLV-1 sero-negative samples and MOT cell line (infected with HTLV-2) amplified only the RPPH1 gene by our method, presenting 100% specificity. 85 Samples confirmed positive or indeterminate by LIA were performed by established qPCR assay and WB. 90.0% (27/30) of LIA-HTLV-1-positive, 33% (2/6) of LIA-untypeable and 2% (1/49) of LIA-indeterminate samples were defined as qPCR-positive. The median PVL of LIA-positive samples (n = 27, 1.780 copies/100 cells) was much higher than that of LIA-untypeable and (n = 2, 0.271 copies/100 cells) indeterminate samples (n = 1, 0.017 copies/ 100 cells). Additionally, compared to WB, the duplex qPCR verified more positive samples, demonstrating a better sensitivity. Conclusion The duplex qPCR developed here with high sensitivity, good specificity and reproducibility could accurately and quantitatively detect the HTLV-1 PVLs, which can be used to confirm the initial reactive samples for an improved cost/benefit ratio as well as to monitor the clinical progression and efficacy of therapy in patients with HTLV-1 related disease.

Potato virus Y (PVY) is a deadly environmental constraint that damages productivity of potato (Solanum tuberosum) around the globe. One of the major challenges is to develop resistance against PVY. Emerging clustered regularly short palindromic repeat (CRISPR)/Cas systems have the potential to develop resistance against PVY. In the current research, CRISPR-Cas13 has been exploited to target multiple strains of PVYN, PVYO, and PVYNTN. Multiple genes PI, HC-Pro, P3, Cl1, Cl2, and VPg genes of PVY were targeted by CRISPR/Cas13a. Multiplex gRNA cassettes were developed on the conserved regions of the PVY-genes. Three independent CRISPR/Cas13 transgenic potato lines were developed by applying an optimized concentration of trans-ribo zeatin and indole acetic acid at callus development, rooting, and shooting growth stages. The level of resistance in transgenic plants was confirmed through double-antibody sandwich enzyme-linked immunosorbent assay and real-time quantitative PCR. Our results have shown that efficiency of PVY inhibition was positively correlated with the Cas13a/sgRNA expression. Finding provides the specific functionality of Cas13 with specific gRNA cassette and engineering the potential resistance in potato crop against multiple strains of PVY.