Single-copy Loss of Rho Guanine Nucleotide Exchange Factor 10 ( arhgef10) Causes Locomotor Abnormalities in Zebrafish Larvae / 生物医学与环境科学（英文）

OBJECTIVE@#To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation.@*METHODS@#Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 -/-, arhgef10 +/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively.@*RESULTS@#WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 -/- zebrafish had more severe symptoms than arhgef10 +/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.@*CONCLUSION@#Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.

Quantitative Expression of RNA from Frozen Organs and Formaldehyde-fixed and Paraffin-embedded Tissues / 法医学杂志

Objective Quantitative analysis and comparison of the expression of ribonucleic acid （RNA） from frozen organs and formaldehyde-fixed and paraffin-embedded （FFPE） tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction （RT-qPCR） technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase （GAPDH） and β-actin （ACTB） with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA （5S rRNA） achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs （miRNAs）, GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.

Inter-laboratory ring trial to evaluate real-time reverse transcription polymerase chain reaction methods used for detection of infectious pancreatic necrosis virus in Chile

Background: Infectious Pancreatic Necrosis Virus (IPNV) is the etiological agent of a highly contagious disease that affects salmonids. In Chile, the second worldwide salmon producer, IPNV causes great economic loss and is one of the most frequently detected pathogens. Due to its high level of persistence and the lack of information about the efficiency of its diagnostic techniques, the National Reference Laboratory (NRL) for IPNV in Chile performed the first inter-laboratory ring trial, to evaluate the sensitivity, specificity and repeatability of the qRT-PCR detection methods used in the country. Results: Results showed 100% in sensitivity and specificity in most of the laboratories. Only three of the twelve participant laboratories presented problems in sensitivity and one in specificity. Problems in specificity (false positives) were most likely caused by cross contamination of the samples, while errors in sensitivity (false negatives) were due to detection problems of the least concentrated viral sample. Regarding repeatability, many of the laboratories presented great dispersion of the results (Ct values) for replicate samples over the three days of the trial. Moreover, large differences in the Ct values for each sample were detected among all the laboratories. Conclusions: Overall, the ring trial showed high values of sensitivity and specificity, with some problems of repeatability and inter-laboratory variability. This last issue needs to be addressed in order to allow harmonized diagnostic of IPNV within the country. We recommend the use of the NRL methods as validated and reliable qRT-PCR protocols for the detection of IPNV.

Standardization and evaluation of a duplex real-time quantitative PCR for the detection of Leishmania infantum DNA: a sample quality control approach

Abstract INTRODUCTION: Molecular techniques have been shown to be alternative methods for the accurate detection of infectious and parasitic diseases, such as the leishmaniases. The present study describes the optimization and evaluation of a duplex real-time quantitative PCR (qPCR) protocol developed for the simultaneous detection of Leishmania infantum DNA and sample quality control. METHODS: After preliminary tests with the newly designed TaqMan® probes for the two targets ( L. infantum and glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene), the duplex qPCR protocol was optimized. For the evaluation of the standardized protocol, human blood samples were tested (n=68) and the results were compared to those obtained by reference diagnostic techniques. Statistical analyses included percentage agreement and the Kappa ( k ) coefficient. RESULTS: The detection limit of L. infantum DNA reached 2x10 2 fg (corresponding to ~1 parasite) per µL of blood (ε: 93.9%). The percentage agreement obtained between the duplex VL qPCR and the reference techniques was individually obtained as follows: molecular: 88.3% ( k =0.666; 95% CI 0.437-0.894, good), and serological: 81.7% ( k =0.411; 95% CI 0.125-0.697, moderate). Between the reference techniques, the percentage agreement was 86.7% ( k =0.586; 95% CI 0.332-0.840, moderate). CONCLUSIONS: The new duplex VL qPCR protocol indicated good potential for the accurate, fast, and reliable detection of L. infantum DNA, when applied as a complement to the classical diagnostic tools already available, especially in health or research reference centers.

Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis

Abstract The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host–pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis.

An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

Estandarización de un método de detección molecular del virus influenza (H5N1) de alta patogenicidad / Standardization of a molecular detection method of highly pathogenic avian influenza virus (H5N1)

El virus de la influenza aviar H5N1 de alta patogenicidad mantiene el alerta mundial debido a su potencial zoonótico y pandémico. Surge entonces la necesidad de contar con herramientas para la detección temprana y de esta forma reducir el impacto potencial a la salud humana y animal. En este estudio se estandarizó un método de detección molecular de los genes de la matriz (M), hemaglutinina (H5) y neuraminidasa (N1) del virus de la influenza aviar H5N1 de alta patogenicidad de linaje asiático, mediante transcripción-reversa y reacción en cadena de la polimerasa en tiempo real (RRT-PCR). A partir de un ARN viral de referencia cepa A/Vietnam/1203/2004 (H5N1) se construyeron controles positivos mediante clonación de productos de PCR. Los estándares de naturaleza plasmídica se emplearon en la obtención de curvas estándar para determinar los límites de detección de la técnica. La sensibilidad observada para todos los genes analizados fue de 10² copias de ADN/μL. Las curvas mostraron una eficiencia superior al 90%, y R²>0,99. Este método puede ser útil en las campañas de monitoreo del virus en aves migratorias, así como para el tamizaje de muestras clínicas de humanos, en una emergencia de salud.

Highly pathogenic avian influenza virus (HPAI) H5N1 is a global threat due to its zoonotic and pandemic potential. Then, concern arises and the need to have early detection tools to minimize the impact on human and animal health. In this work, a molecular detection method was implemented to detect matrix (M), hemagglutinin (H5) and neuraminidase (N1) genes of HPAI avian influenza virus H5N1, based on real time RT-PCR (RRT-PCR). Positive controls were constructed from reference RNA viral A/Vietnam/1203/2004 (H5N1), cloned into plasmidic vectors and sequenced. Assay detection sensitivity was assessed with standard curves for each gene. Assay sensitivity was 10² DNA copies/μl in all cases. Curves showed amplification efficiency higher than 90% and R²>0.99. This method could be useful for bird monitoring campaigns and as a screening procedure for clinical samples.