Reverse transcription-PCR Amplification Research Articles

Point-of-care test of blood Plasmodium RNA within a Pasteur pipette using a novel isothermal amplification without nucleic acid purification

BackgroundResource-limited regions face a greater burden of infectious diseases due to limited access to molecular tests, complicating timely diagnosis and management. Current molecular point-of-care tests (POCTs) either come with high costs or lack adequate sensitivity and specificity. To facilitate better prevention and control of infectious diseases in underserved areas, we seek to address the need for molecular POCTs that better align with the World Health Organization (WHO)’s ASSURED criteria—Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable to end users.MethodsA novel molecular POCT, Pasteur Pipette-assisted isothermal probe amplification (pp-IPA), was developed for malaria detection. Without any microfluidics, this method captures Plasmodium 18S rRNA in a modified Pasteur pipette using tailed genus-specific probes. After washing, the bound tailed probes are ligated to form a template for subsequent novel isothermal probe amplification using a pair of generic primers, bypassing nucleic acid extraction and reverse transcription. The method was assessed using cultured Plasmodium and compared with real-time quantitative reverse transcription PCR (RT-qPCR) or reverse transcription loop-mediated isothermal amplification (RT-LAMP) in clinical blood samples.ResultsThe entire assay is completed in 60–80 min with minimal hands-on time, using only a Pasteur pipette and a water bath. The pp-IPA’s analytical sensitivity is 1.28 × 10–4 parasites/μl, with 100% specificity against various blood-borne pathogens causing malaria-like symptoms. Additionally, pp-IPA needs only liquid-transfer skill for operation and the cost is around USD 0.25 per test, making it at least 300 times lower than mainstream POCT platforms.ConclusionsDesigned to improve the accessibility of molecular detection in resource-limited settings, pp-IPA’s simplicity, affordability, high sensitivity/specificity, and minimal equipment requirements make it a promising point-of-care pathogen identification tool in resource-constrained regions.Graphical

Development of advanced control material for reverse transcription-mediated bacterial nucleic acid amplification tests.

Detection of bacterial RNA by nucleic acid amplification tests (NAATs), such as reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), offers distinct advantages over DNA-based methods. However, such assays also present challenges in ascertaining positive and internal control material that can reliably monitor success over all phases of testing (bacterial lysis, nucleic acid recovery, reverse transcription, amplification, and signal detection): since they are unable to distinguish between amplification of bacterial RNA transcripts and the DNA templates that encode them, using intact organisms as controls can inform cell lysis but not successful detection of RNA. We developed a control strategy for RNA-based bacterial NAATs that allows ready discrimination of RNA from DNA templates using self-splicing bacterial introns, such that those nucleic acids ultimately encode different sequences. We engineered two vectors encoding synthetic transgenes based on this principle, one that is active in the Gram-negative bacterium Escherichia coli and one that functions in both E. coli and the Gram-positive organism Staphylococcus aureus. We subsequently designed RT-LAMP assays that either target RNA and DNA from transgenic organisms or target RNA exclusively and demonstrated the specificity of amplification using purified nucleic acids. Using multiplex fluorescent RT-LAMP of heat-lysed specimens, we showed the practicality of deploying such transgenic organisms as an internal control to ascertain sample integrity and assay performance during clinical diagnostic testing. Our approach has broad utility for RNA-based bacterial NAATs, especially point-of-care assays and other applications where nucleic acids are nonspecifically liberated for testing.

Comparison of 4 laboratory tests for the detection of bovine rabies viral infection in Paraguay: fluorescent antibody test, rapid detection test, histologic lesions, and RT-PCR.

Rabies virus (RABV; Lyssavirus rabies) is a neurotropic virus that can be transmitted to mammals by the hematophagous bat Desmodus rotundus. An accurate, accessible method for the detection of RABV in cattle is necessary in Paraguay; thus, we evaluated the detection of RABV using 4 techniques: fluorescent antibody test (FAT), immunochromatography rapid detection test (RDT; Anigen Rapid Rabies Ag test kit; Bionote), a reverse-transcription PCR (RT-PCR) assay, and histologic lesions in different portions of the CNS of 49 Paraguayan cattle to determine the most sensitive and specific technique. By FAT and RDT, 15 of 49 (31%) samples were positive. By RT-PCR amplification of N and G genes, 13 of 49 (27%) and 12 of 49 (25%) were positive, respectively. RDT had high agreement with FAT (kappa = 1); sensitivity was 100% (95% CI: 97-100%) and specificity was 100% (95% CI: 99-100%). The amplification of the N and G genes resulted in substantial agreement (kappa of 0.9 and 0.8, respectively) compared with FAT, and the sensitivity and specificity of the N gene were 87% (95% CI: 66-100%) and 100% (95% CI: 98-100%), respectively, and those of the G gene were 80% (95% CI: 56-100%) and 100% (95% CI: 98-100%), respectively. Histologic lesions observed were lymphoplasmacytic meningoencephalitis, gliosis, and neuronophagia. The agreement observed between the FAT and RDT tests suggests that RDT is an accurate tool for the detection of RABV. Histopathology can be used to confirm lesions caused by RABV and to rule out other conditions; the RT-PCR assay is useful for molecular epidemiology studies.

Distribution of Wheat-Infecting Viruses and Genetic Variability of Wheat Streak Mosaic Virus and Barley Stripe Mosaic Virus in Kazakhstan.

Wheat is an essential cereal crop for the economy and food safety of Kazakhstan. In the present work, a screening of wheat and barley from different regions of Kazakhstan was conducted using newly developed specific primers for reverse transcription PCR and loop-mediated isothermal amplification (LAMP) assays. In total, 82 and 19 of 256 samples of wheat and barley tested positive for wheat streak mosaic virus (WSMV) and barley stripe mosaic virus (BSMV), respectively. A phylogenetic analysis using two independent methods revealed that most of the analyzed isolates had a European origin. Molecular data on the distribution and diversity of cereal viruses in Kazakhstan were obtained for the first time and will help lay a foundation for the implementation of genetics and genomics in wheat phyto-epidemiology in the country.

A novel splice variant of the human MSTN gene encodes a myostatin-specific myostatin inhibitor.

Myostatin, encoded by the MSTN gene comprising 3 exons, is a potent negative regulator of skeletal muscle growth. Although a variety of myostatin inhibitors have been invented for increasing muscle mass in muscle wasting diseases, no effective inhibitor is currently available for clinical use. Myostatin isoforms in several animals have been reported to inhibit myostatin, but an isoform has never been identified for the human MSTN gene, a conserved gene among animals. Here, a splice variant of the human MSTN gene was explored. Transcripts and proteins were analysed by reverse transcription-PCR amplification and western blotting, respectively. Proteins were expressed from expression plasmid. Myostatin signalling was assayed by the SMAD-responsive luciferase activity. Cell proliferation was assayed by the Cell Counting Kit-8 (CCK-8) assay and cell counting. Cell cycle was analysed by the FastFUCCI system. Reverse transcription-PCR amplification of the full-length MSTN transcript in CRL-2061 rhabdomyosarcoma cells revealed two bands consisting of a thick expected-size product and a thin additional small-size product. Sequencing of the small-size product showed a 963-bp deletion in the 5' end of exon 3, creating exon 3s, which contained unusual splice acceptor TG dinucleotides. The novel variant was identified in other human cell lines, although it was not identified in skeletal muscle. The 251-amino acid isoform encoded by the novel variant (myostatin-b) was identified in CRL-2061 rhabdomyosarcoma cells. Transfection of a myostatin-b expression plasmid into CRL-2061 and myoblast cells inhibited endogenous myostatin signalling (44%, P<0.001 and 63%, P<0.001, respectively). Furthermore, myostatin-b inhibited myostatin signalling induced by recombinant myostatin (68.8%, P<0.001). In remarkable contrast, myostatin-b did not inhibit the myostatin signalling induced by recombinant growth differentiation factor 11 (9.2%, P=0.70), transforming growth factor β (+3.1%, P=0.83) or activin A (+1.1%, P=0.96). These results indicate the myostatin-specific inhibitory effect of myostatin-b. Notably, the expression of myostatin-b in myoblasts significantly enhanced cell proliferation higher than the mock-transfected cells by the CCK-8 and direct cell counting assays (60%, P<0.05 and 39%, P<0.05, respectively). Myostatin-b increased the percentage of S-phase cells significantly higher than that of the mock-transfected cells (53% vs. 80%, P<0.05). We cloned a novel human MSTN variant produced by unorthodox splicing. The variant encoded a novel myostatin isoform, myostatin-b, that inhibited myostatin signalling by myostatin-specific manner and enhanced myoblast proliferation by shifting cell cycle. Myostatin-b, which has myostatin-specific inhibitory activity, could be developed as a natural myostatin inhibitor.

Development of a reagent kit for the qualitative detection of SARS-CoV-2 virus RNA in naso- and oropharyngeal swabs by real-time RT-PCR.

The coronavirus infection continues to spread around the world. In this regard, the purpose of this work was: to develop a set of reagents for the qualitative detection of SARS-CoV-2 virus RNA. The set was developed by CJSC «Ecolab», 20 positive samples were used to develop the kit. The research method consisted of several stages: isolation of SARS-CoV-2 RNA, RNA reverse transcription reaction and PCR amplification of cDNA with simultaneous detection of the result in real time. The main characteristics of the kit: analytical sensitivity - 100%, specificity - 100%, accuracy - 100%. Thus, our method for diagnosing a new coronavirus infection based on real-time RT-PCR makes it possible to qualitatively and quickly detect betacoronavirus RNA in clinical material from patients and healthy individuals with suspected coronavirus infection and other symptoms of SARS.

RNAi-mediated knockdown of arginine kinase genes leads to high mortality and negatively affect reproduction and blood-feeding behavior of Culex pipiens pallens.

Arginine kinase (AK) is one of the crucial enzymes involved in energy metabolism in invertebrates, and has been proposed as the target for RNA interference (RNAi)-based control of agricultural insect pests. While there is only one AK gene in most insects, two AK genes were identified in Culex pipiens pallens, the primary vector of lymphatic filariasis and epidemic encephalitis. The full-length cDNA sequences of CpAK1 and CpAK2 genes were obtained by reverse transcription PCR(RT-PCR) and rapid amplification of cDNA ends (RACE). The expression levels of CpAK1 and CpAK2 in different developmental stages and tissues were detected by reverse transcription quantitative PCR (RT-qPCR). The role of CpAK1 and CpAK2 in the reproduction and blood feeding behavior was analyzed using RNA interference (RNAi). Full-length cDNAs of CpAK1 and CpAK2 were isolated from Cx. pipiens pallens. Analysis of the expression pattern revealed that the mRNA level of CpAK1 was significantly higher than CpAK2 in all development stages and tissues examined, and the expressions of both CpAK1 and CpAK2 were upregulated in response to blood feeding. The co-knockdown of CpAK1 and CpAK2 mediated by RNAi led to high mortality (74.3%) of adult female mosquitoes and decreased hatchability (59.9%). Remarkably, the blood feeding rate and the engorgement rate of the female mosquitoes were negatively affected by co-injection of dsRNAs targeting CpAK1 and CpAK2. CpAK1 and CpAK2 were detected in all developmental stages and tissues, but showed divergence in expression level. RNAi-mediated knockdown of AK genes leads to high mortality and negatively affect blood-feeding behavior of Cx. pipiens pallens, suggesting that AK could be used for the target of RNAi-based mosquito control in the future.

Abstract 4104: Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology

Abstract Introduction: The FDA has approved 11 therapies for non-small cell lung cancer (NSCLC) patients diagnosed with gene fusions. Importantly, these life-saving targeted therapies remain underutilized due to a lack of cost-effective, comprehensive, and simple-to-use technologies for gene fusion detection. Our previous work using ASPYRE technology (allele-specific pyrophosphorolysis reaction; Silva et al, 2020) demonstrated detection of somatic variants at single-molecule level from DNA extracted from tumor tissue. However, DNA-based fusion detection requires tiling across large introns, which limits sensitivity particularly if breakpoints are located in repetitive regions of the genome. Here, we show that ASPYRE technology can also be utilized for highly sensitive detection of RNA fusions, scaling to over 30 fusion targets covering the main 3’ fusion partners (ALK, ROS1, RET, NTRK1, NTRK3) and from a single reverse transcription PCR (RT-PCR) amplification reaction. Methods: The ASPYRE assay consists of four sequential enzymatic stages: RT-PCR; enzymatic clean-up; combined exonuclease digestion, hybridization, pyrophosphorolysis and ligation; rolling circle isothermal amplification and detection. All targets are detected across two reaction wells using four channels on a standard real-time PCR instrument. A panel of synthetic RNA oligonucleotides comprising gene fusion boundaries was spiked into a background pool of lung-RNA, quantified using digital PCR (dPCR) and compared against a commercial reference standard to validate its suitability for assessing assay performance. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) lung tissue from healthy donors to determine assay specificity, and from FFPE lung tissue from NSCLC patients with known fusions to exemplify end-to-end sample analysis. Results: We demonstrate equivalence of our in-house reference samples to commercial reference standards. Using serial dilutions of input RNA standards, we show that detection by ASPYRE is consistent with detection of single molecules as input for common ROS1, ALK, RET and NTRK3 fusions. In addition, ASPYRE was able to detect previously confirmed ROS1 and ALK fusions and MET exon skipping variants in FFPE samples. ASPYRE had high specificity, correctly confirming the absence of RNA fusions and exon skipping variants in healthy control FFPE samples. Conclusions: ASPYRE approached single-molecule sensitivity for over 30 RNA fusion targets in a single reaction. The assay required only 1 ng RNA input, with a turn-around time of &amp;lt;4 hours from extracted RNA to result, opening the door to routine detection of RNA fusions from limited specimens. The simplicity and ease of use of ASPYRE, combined with its rapid turnaround time and analytical performance characteristics, provides a new tool for rapid and comprehensive treatment selection in NSCLC. Citation Format: Eleanor Gray, Justyna Mordaka, Efthimia Christoforou, Kristine von Bargen, Nicola Potts, Rebecca Palmer, Barnaby Balmforth. Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4104.

Cloning and functional analysis of the phenylalaninammo-nialyase gene from Rhododendron fortunei

Phenylalaninammo-nialyase (PAL) is a key enzyme in the synthesis of methyl benzoate - a plant aroma compound. In order to understand the function of this enzyme in the formation of fragrance in the scented Rhododendron species-Rhododendron fortunei, we cloned a gene encoding this enzyme and subsequently examined the gene expression patterns and the profile of enzyme activity during development in various tissues. The full length of RhPAL gene was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The expression levels of RhPAL gene were measured by real-time quantitative reverse transcription PCR (qRT-PCR) and the amount of phenylalanine and cinnamic acid were assayed with LC-MS. The results showed that the ORF sequence of RhPAL gene amplified from the cDNA templates of flower buds had 2 145 bp, encoding 715 amino acids, and shared 90% homology to the PAL amino acid sequences from other species. qRT-PCR analysis showed that the expression of RhPAL in petals during flowering kept in rising even until the flowers wilted. The expression of RhPAL in pistil was much higher than that in stamen, while the expression in the younger leaves was higher than in old leaves. However, the expression level was relatively lower in petal and stamen compared to that in leaves. We also measured the PAL activity by Enzyme-linked immuno sorbent assay in the petals of flowers at different flowering stages. The results showed that PAL activity reached the highest at the bud stage and then decreased gradually to the lowest when the flowers wilted, which followed a similar trend in the emission of the flower fragrance. The phenylalanine and cinnamic acid contents measured by LC-MS were highly correlated to the expression level of RhPAL in various tissues and at different flowering stages, implying that RhPAL plays an important role in the formation of the flower fragrance. This work may facilitate the breeding and improvement of new fragrant Rhododendron cultivars.

Universal Primers for Rapid Detection of Six Pospiviroids in Solanaceae Plants Using One-Step Reverse-Transcription PCR and Reverse-Transcription Loop-Mediated Isothermal Amplification.

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are listed as quarantine pathogens in many countries. Among them, columnea latent viroid, pepper chat fruit viroid, potato spindle tuber viroid, tomato apical stunt viroid, tomato chlorotic dwarf viroid, and tomato planta macho viroid are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from 1 fg to 10 ng, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR, but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate the ability to screen a large number of solanaceous plants and seeds intended for import and export.

Watermelon crinkle leaf-associated virus 1 and watermelon crinkle leaf-associated virus 2 have a bipartite genome with molecular signatures typical of the members of the genus Coguvirus (family Phenuiviridae).

Watermelon crinkle leaf-associated virus 1 and watermelon crinkle leaf-associated virus 2 (WCLaV-1 and WCLaV-2), two unclassified members of the order Bunyavirales, are phylogenetically related to members of the genus Coguvirus (family Phenuiviridae). The genome of both viruses was reported previously to be composed of three RNA segments. However, the terminal sequences of two genomic RNA segments, namely those encoding the putative movement protein (MP) and the nucleocapsid (NP) protein, remained undetermined. High-throughput sequencing of total RNA and small RNA preparations, combined with reverse transcription PCR amplification followed by sequencing, revealed that the WCLaV-1 and WCLaV-2 possess a bipartite genome consisting of a negative-sense RNA1, encoding the RNA-dependent RNA polymerase, and an ambisense RNA2, encoding the putative movement (MP) and nucleocapsid (NP) proteins. The two open reading frames of RNA2 are in opposite orientations and are separated by a long AU-rich intergenic region (IR) that may assume a hairpin conformation. RNA1 and RNA2 of both viruses share almost identical 5' and 3' termini, which are complementary to each other up to 20 nt. This genome organization is typical of members of the genus Coguvirus, with which WCLaV-1 and WCLaV-2 also share similar terminal 5' and 3' sequences of RNA1 and RNA2. These molecular features, together with phylogenetic reconstructions support the classification of WCLaV-1 and WCLaV2 as members of two new species in the genus Coguvirus.

Molecular cloning and functional studies on magang goose toll-like receptor 5

Disease outbreaks heavily impact the economic viability of animal industries. Little is known about the mechanisms of immune system-related diseases in geese. Toll-like receptors (TLRs) play a major role in the anti-inflammatory immunity process in most animal species, but they have not been studied in the Magang goose. To elucidate the role of TLRs, reverse transcription polymerase chain reaction (RT-PCR) and PCR amplification of cDNA ends (Smart RACE) were used to clone the Magang goose TLR5 gene (mgTLR5). The full-length cDNA of mgTLR5 was 2967 bp in length, including a 5′-terminal untranslated region (UTR) of 215 bp, a 3′-terminal UTR of 384 bp, and an open reading frame of 2583 bp that encodes a protein of 860 amino acids. Structurally, mgTLR5 has a toll/interleukin-receptor (TIR) domain, a transmembrane domain, and seven leucine-rich repeats (LRRs) domains. Homology alignment of TLR5 and its TIR domains with other species revealed that mgTLR5 shared 98 % and 81.3 % of sequence similarity with white goose TLR5 and chicken TLR5, respectively. Quantitative RT-PCR showed that the mgTLR5 gene of the goose is widely expressed in all tested tissues, with the highest expression in the kidney and spleen. The increase in NF-κB promoter activity stimulated by flagellin was dependent on mgTLR5 expression in 293 T cells. Salmonella pullorum and flagellin significantly upregulated the expression of TLR5, IL-8, and IL-1 mRNA in peripheral blood mononucleotide cells of Magang goose cultured in vitro. Stimulation by S. pullorum for 24 h upregulated mgTLR5 expression in the cecum and kidney. We conclude that Magang goose TLR5 is a functional TLR5 homologue of the protein in other species and plays an important role in bacterial recognition.

Rapid High-Throughput Whole-Genome Sequencing of SARS-CoV-2 by Using One-Step Reverse Transcription-PCR Amplification with an Integrated Microfluidic System and Next-Generation Sequencing.

The long-lasting global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Here, we report a simple and efficient workflow for whole-genome sequencing utilizing one-step reverse transcription-PCR (RT-PCR) amplification on a microfluidic platform, followed by MiSeq amplicon sequencing. The method uses Fluidigm integrated fluidic circuit (IFC) and instruments to amplify 48 samples with 39 pairs of primers, including 35 custom-designed primer pairs and four additional primer pairs from the ARTIC network protocol v3. Application of this method on RNA samples from both viral isolates and clinical specimens demonstrates robustness and efficiency in obtaining the full genome sequence of SARS-CoV-2.

Molecular Cloning, Primary Sequence Characterization and Phylogentic Analysis of Cathepsin B cysteine from Sheep (Ovis aries) Ovarian

Background: CathepsinB (CTSB) is a lysosomal cysteine protease which has been implicated in a variety of physiological and pathological processes. However, the sequence and structure characteristics of sheep CTSB gene are still unclear, which severely restricts the further study about genetic variation, molecular evolution.Methods: Therefore the coding sequence (CDS) of sheep CTSB gene from ovary was cloned by reverse transcription PCR (RT-PCR) amplification and regular sequencing technology. The characteristics of nucleotide and amino acid sequence of CTSB gene were analyzed through bioinformatics and the phylogenetic tree was constructed employing the Bayesian inference based on amino acid sequence of different species in this study.Result: A complete coding sequence of 1008 bp of CTSB was obtained from ovary, which encoded 335 amino acids. Functional prediction of CTSB protein suggested that containing five conserved sequence motif sites in which the site of amino acids between 158 and 205 was the most conserved region. Secondary structure analysis showed that á-helices were the major structure at the N-terminus, while random coils were the major structure at the C-terminus. Additionally, the motif II in the random coils, in which two active sites of His278 and Asn298 were located therein, was more conserved. The high similarity of CTSB amino acids alignment between sheep and other species recommended that CTSB was highly conservative in the evolutionary process.

Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene.

Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splicing assay easier, a ready-made minigene (FMv2) amenable to quantitative splicing analysis by fluorescence microscopy was constructed. FMv2 was designed to encode two fluorescence proteins namely, mCherry, a transfection marker and split eGFP, a marker of splicing reaction. The split eGFP was intervened by an artificial intron containing a multicloning site sequence. Expectedly, FMv2 transfected HeLa cells produced not only red mCherry but also green eGFP signals. Transfection of FMv2CD44v8, a modified clone of FMv2 carrying an insertion of CD44 exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested against exon v8, ASO#14 produced the highest index of green signal positive cells. Hence, ASO#14 was the most efficient exon v8 skippable ASO. Notably, the well containing ASO#14 was clearly identified among the 96 wells containing randomly added ASOs, enabling high throughput screening. A ready-made FMv2 is expected to contribute to identify exon skippable ASOs.

Mechanism of Reproductive Effect of Acetyl acetate‐methanol Fraction of Adansonia digitata (Linn) Root Bark Extract in Female Wistar Rats

The aim of this work was to identify the exclusive components of Acetyl acetate‐methanol fraction (AAMF) of Adansonia digitata (Linn) root bark extract and to elucidate its mechanism of action in modulating hormone signaling. Gas chromatography and Mass spectrometry was done to identify the compounds present in fractions of A. digitata (Linn) root bark extract. Thirty female Wistar rats had their estrous cycles synchronized and were randomly distributed into three groups of ten rats each. Each group received 0, 150, and 300mg/kg body weight of AAMF per os from the day observed as proestrus by vaginal cytology to day‐seven. On day‐one, day‐four, day‐five, day‐six and day‐seven, blood was collected from rats in each group. Blood collected was analyzed for estrogen, progesterone and follicle stimulating hormone levels. Uterus and ovary were processed for histopathology and immunohistochemistry to study the regulation of estrogen and progesterone receptor proteins. Where a receptor protein was regulated, further investigation was done to determine its effect on the gene expression of that receptor in endometrial stromal cells through RNA extraction, OneStep reverse transcription‐pCR amplification, Agarose gel electrophoresis and quantification of pCR product. Results showed that AAMF uniquely contained 15.25% of oleic acid and caused mass atresia of antral follicles at proestrus. AAMF treated rats had significantly (p&lt;0.05) lowered serum estradiol levels at proestrus when compared to control rats and had up‐regulated estrogen receptor beta proteins in endometrium and downregulated estrogen receptor alpha (ERα) gene expression in endometrial cells. It was concluded that the oleic acid component of AAMF may have lowered estrogen level by inhibiting aromatase and hydroxysteroid 17 beta dehydrogenase enzyme activities in estrogen steroidogenesis. Also, oleic acid may have induced overexpression of Tumour Suppressor Factor (P53) which promotes follicular apoptosis and indirectly downregulates ERα when p53 activities become exacerbated. It is recommended that AAMF be investigated as a possible therapy for some ERα sensitive clinical conditions like cancer.

Paper Lateral Flow Biosensor for Nodavirus Reverse Transcribed RNA Detection.

Paper nanobiosensors have been established as an excellent platform for analysis of veterinary and human pathogens causing various diseases. Especially, lateral flow assays or biosensors ideal for sensitive, rapid, robust and accurate analysis in laboratory setups and on-site analysis. Viral RNA detection is of great importance for public health as well as animal health protection. In that aspect, the present protocol focuses on the development of functionalized gold nanoparticle-based lateral flow biosensor for fish nervous necrosis virus (Nodavirus) nucleic acids detection. Total viral RNA, isolated from fish samples was subjected to reverse transcription PCR amplification and the amplification products were mixed with specific oligonucleotide probe. A red test line was formed when nodavirus product was present. The proposed assay has great implications on basic research since it eliminates the need for time-consuming, cumbersome electrophoresis protocols and could be adjusted for use on the site of fish culture by fish farmers. Disease monitoring by such bioanalytical platforms without time consuming and costly procedures would have great impact on the aquaculture and environmental safety.

Complete genomic sequence of crow-dipper mosaic-associated virus, a novel macluravirus infecting Pinellia ternata.

A new macluravirus infecting Pinellia ternata in China was identified by high-throughput sequencing (HTS) and tentatively named "crow-dipper mosaic-associated virus" (CrdMV). The complete genome sequence of CrdMV was determined by reverse transcription (RT) PCR and rapid amplification of cDNA ends (RACE) PCR. The genomic RNA of CrdMV consists of 8,454 nucleotides (nt), excluding the poly(A) tail at the 3' end. CrdMV has a genomic structure typical of macluraviruses, with large open reading frame encoding a polyprotein of 2,696 amino acids (aa). CrdMV shares 54.40%-59.37% nt sequence identity at the genome sequence level, 48.00%-58.58% aa sequence identity, at the polyprotein sequence level and 37.27%-49.22% aa sequence identity at the CP sequence level with other members of the genus Macluravirus. These values are well below the species demarcation threshold for the family Potyviridae. Phylogenetic analysis based on the amino acid sequences of polyproteins confirmed that CrdMV clusters closely with broad-leafed dock virus A (BDVA, GenBank accession no. KU053507). These results suggest that CrdMV should be considered a distinct member of the genus Macluravirus.

Duplication and soldier-specific expression of geranylgeranyl diphosphate synthase genes in a nasute termite Nasutitermes takasagoensis

In the evolutionarily-derived termite subfamily Nasutitermitinae (family Termitidae), soldiers defend their nestmates by discharging polycyclic diterpenes from a head projection called the “nasus.” The diterpenes are synthesised in the frontal gland from the precursor geranylgeranyl diphosphate (GGPP), which is generally used for post-translational modification of proteins in animals. In this study, we constructed a comprehensive gene catalogue to search for genes involved in the diterpene biosynthesis by assembling RNA sequencing reads of Nasutitermes takasagoensis, identifying eight gene copies for GGPP synthase (GGPPS). The number of gene copies is much larger in contrast to other related insects. Gene cloning by reverse transcription-PCR and rapid amplification of cDNA ends confirmed that seven GGPPS genes (NtGGPPS1 to NtGGPPS7) have highly variable untranslated regions. Molecular phylogenetic analysis showed that the NtGGPPS7 gene was grouped with homologs obtained from ancestral termites that have only a single copy of the gene, and the NtGGPPS6 gene was grouped with homologs obtained from a basal lineage of termitids, in which soldiers do not synthesise diterpenes. As the sister group to this clade, furthermore, a monophyletic clade included all the other NtGGPPS genes (NtGGPPS1 to NtGGPPS5). Expression analyses revealed that NtGGPPS7 gene was expressed in all the examined castes and tissues, whereas all the other genes were expressed only in the soldier head. These results suggest that gene duplication followed by subfunctionalisation of the GGPPS genes might have accompanied the evolution of chemical defence in the nasute termite lineage.

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and dengue viruses in mosquitoes

BackgroundThe global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed.MethodsWe present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations (“artificially expanded genetic information systems”, AEGIS, and “self-avoiding molecular recognition systems”, SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides).ResultsThe diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3–4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels.ConclusionsOur results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission.