A Panel of Real-Time PCR Assays for the Direct Detection of All of the Xylella fastidiosa Subspecies.

The bacterial plant pathogen Xylella fastidiosa causes disease in hundreds of plant species worldwide including many crops, and as such accurate determination of the subspecies of the bacteria is vital to control, containment, and eradication measures. Conventional methods to determine the subspecies of X. fastidiosa rely on time consuming multilocus sequence typing (MLST), a laborious multistage process. This chapter provides a rapid alternative to MLST utilizing real-time PCR assays to provide highly specific and sensitive detection of the pathogen subspecies. Here we describe the methodology for sampling plant material, performing the DNA extraction and undertaking the real-time PCR assays. This method allows straightforward, robust, reliable, high-throughput, and rapid determination of the X. fastidiosa subspecies.

Accuracy of Real-Time Polymerase Chain Reaction in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is a mild to severe respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The diagnostic accuracy of the Centers for Disease Control and Prevention (CDC)- or World Health Organization (WHO)-recommended real-time PCR (RT-qPCR) primers in clinical practice remains unproven. We conducted a prospective study on the accuracy of RT-qPCR using an in-house-designed primer set (iNP) targeting the nucleocapsid protein as well as various recommended and commercial primers. The accuracy was assessed by culturing or seroconversion. We enrolled 12 confirmed COVID-19 patients with a total of 590 clinical samples. When a cutoff value of the cycle threshold (Ct) was set to 35, RT-qPCRs with WHO RdRp primers and CDC N1, N2, and N3 primers showed sensitivity of 42.1% to 63.2% and specificity of 90.5% to 100% in sputum, and sensitivity of 65.2% to 69.6% and specificity of 65.2% to 69.6% in nasopharyngeal samples. The sensitivity and specificity of iNP RT-qPCR in sputum and nasopharyngeal samples were 94.8%/100% and 69.6%/100%, respectively. Sputum testing had the highest sensitivity, followed by nasopharyngeal testing (P = 0.0193); self-collected saliva samples yielded better characteristics than oropharyngeal samples (P = 0.0032). Our results suggest that iNP RT-qPCR has better sensitivity and specificity than RT-PCR with WHO (P < 0.0001) or CDC (N1: P = 0.0012, N2: P = 0.0013, N3: P = 0.0012) primers. Sputum RT-qPCR analysis has the highest sensitivity, followed by nasopharyngeal, saliva, and oropharyngeal assays. Our study suggests that considerable improvement is needed for the RT-qPCR WHO and CDC primer sets for detecting SARS-CoV-2. IMPORTANCE Numerous research campaigns have addressed the vast majority of clinical and diagnostic specificity and sensitivity of various primer sets of SARS-CoV2 viral detection. Despite the impressive progress made to resolve the pandemic, there is still a need for continuous and active improvement of primers used for diagnosis in clinical practice. Our study significantly exceeds the scale of previously published research on the specificity and sensitivity of different primers comparing with different specimens and is the most comprehensive to date in terms of constant monitoring of primer sets of current usage. Henceforth, our results suggest that sputum samples sensitivity is the highest, followed by nasopharyngeal, saliva, and oropharyngeal samples. The CDC recommends the use of oropharyngeal specimens, leading to certain discrepancy between the guidelines set forth by the CDC and IDSA. We proved that the oropharyngeal samples demonstrated the lowest sensitivity for the detection of SARS-CoV-2.

Development and validation of a multiplex real-time qPCR assay using GMP-grade reagents for leprosy diagnosis.

Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.

A Method for Evaluation of the Level of Circulating Mitochondrial DNA by ND1 and ND2 Genes.

The measurement of the level of mitochondrial DNA (mtDNA) in the blood is a difficult problem due to high variability of mitochondrial genes, deletions in the mitochondrial genome in some pathological conditions, different sources of mtDNA into the bloodstream (mtDNA from tissues, from blood cells, etc.). We designed primers and TaqMan probes for highly conserved regions of the ND1 and ND2 genes outside the mitochondrial deletions "hot zones". For standardizing the technique, the true concentration of low-molecular-weight mtDNA was determined by real-time PCR for two targets: a fragment of the ND2 gene (122 bp) and the ND1 and ND2 genes (1198 bp). The sensitivity and specificity of the developed approach were verified on a DNA pool isolated from the blood plasma of healthy donors of various nationalities. The concentration of low-molecular-weight mtDNA in the blood plasma of two patients with COVID-19 was monitored over two weeks of inpatient treatment. A significant increase in the content of low-molecular-weight mtDNA was observed during the first 5 days after hospitalization, followed by a drop to the level of healthy donors. The developed technique makes it possible to assess the blood level of low-molecular-weight mtDNA regardless of the quality of sampling and makes it possible to standardize this biological marker in a wide range of infectious and non-infectious pathologies.

Evaluation of stable reference genes for qPCR normalization in circadian studies related to lung inflammation and injury in mouse model.

Circadian rhythms have a profound effect on lung function and immune-inflammatory response in chronic airway diseases. Thus, understanding the molecular mechanisms of circadian gene expression of core clock-controlled genes (CCGs) may help better understand how it contributes to the physiology and pathology of lung diseases. Ongoing studies have been analyzing gene expression levels of CCGs in mouse lungs using quantitative real-time PCR (qRT-PCR). However, to date, there are no reports on the most stable reference gene in the mouse lung for circadian studies. Herein, we utilized an acute house dust mite (HDM)-sensitization mouse model to evaluate the stability of 10 reference genes commonly used for qRT-PCR normalization using 5 unique algorithms: GeNorm, NormFinder, BestKeeper, RefFinder and Qbase+. Rn18s was determined as the most stable reference gene across all samples evaluated, and Actb, the least stable reference gene. Furthermore, CircWave analysis showed no diurnal variation in the expression pattern for Rn18s but Actb showed strong diurnal changes in the lungs of both PBS (control) and HDM groups. We demonstrate systematically how using Actb as a housekeeping gene offsets the diurnal expression patterns of the CCGs and leads to statistically significant results which may not be the true reflection of the qRT-PCR analysis.

Comparison of three molecular diagnostic assays for SARS-CoV-2 detection: Evaluation of analytical sensitivity and clinical performance.

BACKGROUND: Currently, SARS-CoV-2 RNA detection using real-time reverse-transcription PCR (rRT-PCR) is the standard diagnostic test for COVID-19 infection. Various rRT-PCR assays are currently used worldwide, targeting different genes of the SARS-CoV-2. Here, we compared the analytical sensitivity and clinical performance (sensitivity and specificity) of Allplex SARS-CoV-2/FluA/FluB/RSV assay (Seegene), Standard M nCoV real-time detection kit (SD Biosensor), and U-TOP COVID-19 detection kit (Seasun Biomaterials) for SARS-CoV-2 detection. METHODS: Two hundred and forty-nine nasopharyngeal swab samples were evaluated to compare the clinical performance of the rRT-PCR assays. For the analytical performance evaluation, two RNA controls with known viral loads-SARS-CoV-2 RNA control and SARS-COV-2 B.1.351 RNA control-were used to investigate the potential impact of SARS-CoV-2 variants, particularly the B.1.351 lineage. RESULTS: Limits of detection ranged from 650 to 1300 copies/ml for rRT-PCR assays, and the mean differences in cycle threshold (Ct ) values of the two RNA controls were within 1.0 for each target in the rRT-PCR assays (0.05-0.73), without any prominent Ct value shift or dropouts in the SARS-COV-2 B.1.351 RNA control. Using the consensus criterion as the reference standard, 89 samples were positive, whereas 160 were negative. The overall clinical performance of rRT-PCR assays was comparable (sensitivity 98.88%-100%; specificity 99.38%-100%), whereas the sensitivities of each target gene were more variable. CONCLUSIONS: The three rRT-PCR assays showed comparable analytical sensitivity and clinical performance. The analytical and clinical sensitivities of each target gene were influenced more by the primer and probe design than the target gene itself.

Improved Detection of Little Cherry Virus-2 Using a Hydrolysis Probe to Manage the Pacific Northwest Little Cherry Disease Epidemic.

Little cherry virus-2 (LChV-2) is a viral pathogen that is reaching epidemic levels in Washington State. This virus is insect vectored and has significant impacts on sweet cherry production. To aid growers in making informed management decisions, we sought to develop a diagnostic assay to better detect isolates of LChV-2 currently found in Washington, allowing more accurate estimations of disease occurrence. This study showed that there were two distinct genotypes of LChV-2 present in Washington State. This information was used to develop an up-to-date reverse transcription real-time quantitative PCR assay, which was then optimized, validated, and compared with four previously published assays of a panel of field samples. This comparison demonstrated that the newly developed assay provided greater sensitivity, accurately detecting <10 copies per reaction and could detect both LChV-2 genotypes. Finally, we examined the effect of potential inhibitors in various tissue types from cherry, finding that young leaf tissue affected sensitivity of detection less than root tissues.

Evaluation of qPCR reference genes for taimen (Hucho taimen) under heat stress.

As a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquaculture research. Understanding the biology of taimen (Hucho taimen) has drawn increasing interest because of its ecological and economic value. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimized for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder. The expression levels of the 10 genes were detected using 240 samples from 48 experimental groups consisting of 40 individuals treated under four heat-stress conditions (18, 20, 22, and 24 °C) for 24 h and 26 °C for 4, 24, 48, and 72 h. Six tissues (blood, heart, brain, gill, skin, and liver) were collected from each individual. Ribosomal protein S29 (RPS29) and ribosomal protein L19 (RPL19) were the most stable genes among all of the samples, whereas 28S ribosomal RNA (28S rRNA), attachment region binding protein (ARBP), and 18S ribosomal RNA (18S rRNA) were the least stable. These results were verified by an expression analysis of taimen heat-stress genes (heat shock protein 60, hsp60, and heat shock protein 70, hsp70). In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions. These results allow the reliable study of gene expression in taimen.

Single-copy Loss of Rho Guanine Nucleotide Exchange Factor 10 ( arhgef10) Causes Locomotor Abnormalities in Zebrafish Larvae.

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.

Comparison of pneumococcal colonization density among healthy children and children with respiratory symptoms using real time PCR (RT-PCR).

BACKGROUND: Nasopharyngeal colonization is considered a necessary step in the initiation of pneumococcal diseases. Real time PCR (RT-PCR) is an alternative approach for the identification and quantification of pneumococci directly from samples. OBJECTIVES: To compare pneumococcal detection rates using culture-based method versus RT-PCR direct detection and to quantify pneumococcal colonization in two study cohorts (healthy children and hospitalized children with respiratory symptoms) using quantitation through RT-PCR. METHODOLOGY: A total of 101 nasopharyngeal swabs (NPS) from healthy children and 183 NPSs from hospitalized children with respiratory symptoms were included in the study. None of the children were vaccinated. All children were between 2 months to 2 years. In parallel to routine culture and identification, a RT-PCR assay targeting the lytA gene was done. RESULTS: Considering all 284 samples tested, colonization rate by conventional culture was 41.2% (n = 117) while positive colonization using RT-PCR was 43.7% (n = 124). The colonization rate detected by RT-PCR in the healthy cohort was 33.7% (n = 34) and it was 49.2% (n = 90) in the hospitalized cohort. It was 37.6% (n = 38) and 43.2% (n = 79) for the two cohorts by culture. The mean Cq value for the healthy cohort is 29.61 (SD 2.85) and 28.93 (SD 3.62) for the hospitalized cohort. With the standard curve obtained from amplifying a dilution series of control DNA, the mean amount of genomic DNA copy numbers detected in children with respiratory symptoms was log10 7.49 (SD 1.07) while it was log10 7.30 (SD 0.23) in healthy children and the difference was not statistically significant. CONCLUSIONS: The overall colonization rate was higher when detected using RT-PCR compared to culture. However, it was lower in the healthy group when detected with RT-PCR compared to culture. Even though there was a higher detection of pneumococcal colonization density in children with respiratory symptoms, this was not significantly higher unlike many previous studies. Therefore, the use of RT-PCR to detect pneumococcal colonization needs further evaluation with careful analysis of interpretation and confounders.

One-step and sequential SARSCOV-2 polymerase chain reaction tests would not work every time.

INTRODUCTION: RT-PCR is widely used as a diagnostic test for the detection of SARS-CoV-2. In this study, we aim to describe the clinical utility of serial PCR testing in the final detection of COVID-19. METHOD: We collected multiple nasopharyngeal swab samples from patients who had negative RT-PCR test on the first day after hospitalization. RT-PCR tests were performed on the second day for all patients with initial negative result. For the patients with secondary negative results on day 2, tertiary RT-PCR tests were performed on day 3 after hospitalization. RESULT: Among 68 patients with initial negative test results, at the end of follow-up, the mortality number was 20 (29.4%). About 33.8% of patients had subsequent positive PCR test results for the second time and 17.4% of the patients who performed third PCR test had positive result. CONCLUSION: Based on this study, serial RT-PCR testing is unlikely to yield additional information.

Log (Lactobacillus crispatus/ Gardnerella vaginalis): a new indicator of diagnosing bacterial vaginosis.

To explore a new marker which can detect bacterial vaginosis (BV) with high sensitivity and specificity quantitatively. According to the Nugent Score, vaginal secretions from study participants were divided into BV, healthy, and BV-intermediate groups. First, we compared the obvious differences and high abundance of bacteria in the three groups using 16S rRNA-sequencing, and screened out candidate markers. Then, quantitative detection of these candidate markers from the three groups was done using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), followed by evaluation of the sensitivity and specificity. Finally, we verified the new markers using clinical cases. Gardnerella vaginalis, Atopobium vaginae, Lactobacillus, Megasphaera were screened out by 16S rRNA-sequencing. RT-qPCR data were transformed and analyzed through ROC curves. PCR results for these bacteria were log-transformed using Lactobacillus crispatus as the numerator and other BV-related bacteria as the denominator. Four new indicators were found. Of these, log L. crispatus/G. vaginalis (L/G) <0 was the best indicator. The sensitivity, specificity, positive predictive value, and negative predictive value of our system were 93.5%, 97.2%, 96.6 and 94.6%, respectively. Combination of data for 16S rRNA-sequencing and RT-qPCR revealed four indicators for BV detection. Of these, log L/G < 0 was the best indicator. Creating a molecular-diagnostic system independent of the Nugent Score for BV could have an important impact on the clinical management of BV.Abbreviation: log L. crispatus/G. vaginalis (logL/G); Bacterial vaginosis (BV); vaginal secretions (VSs); polymerase chain reaction (PCR); rRNA-sequencing (rRNA-seq); real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR); operational taxonomic unit (OTU); non-metric multidimensional scaling (NMDS); receiver operating characteristic (ROC).

Screening and stability analysis of reference genes in fasting caecotrophy model in rabbits.

BACKGROUND: The selection and validation of stably expressed reference genes is key for accurately quantifying the mRNA abundance of genes under different treatments. In the rabbit model of fasting caecotrophy, reports about the selection of stable reference genes are not available. METHODS AND RESULTS: This study aims to screen suitable reference genes in different tissues (including uterus, cecum, and liver) of rabbits between control and fasting caecotrophy groups. RT-qPCR was used to analyze the expression levels of eight commonly used reference genes (including GAPDH, 18S rRNA, B2M, CYP, HPRT1, ß-actin, H2afz, Ywhaz), and RefFinder (including geNorm, NormFinder, and BestKeeper) was used to analyze the expression stability of these reference genes. Our results showed that the most stable reference genes were different in different tissues and treatments. In the control and fasting caecotrophy groups, CYP, GAPDH and HPRT1 were proven to be the top stable reference genes in the uterus, cecum, and liver tissues, respectively. GAPDH and Ywhaz were proven to be the top two stable reference genes among uterus, cecum, and liver in both control and fasting caecotrophy groups. CONCLUSIONS: Our results indicated that the combined analysis of three or more reference genes (GAPDH, HPRT1, and Ywhaz) are recommended to be used for RT-qPCR normalization in the rabbit model of fasting caecotrophy, and that GAPDH is a better choice than the other reference genes for normalizing the relative expression of target genes in different tissues of fasting caecotrophy rabbits.

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.

The use of HRM shifts in qPCR to investigate a much neglected aspect of interference by intracellular nanoparticles.

Genetic molecular studies used to understand potential risks of engineered nanomaterials (ENMs) are incomplete. Intracellular residual ENMs present in biological samples may cause assay interference. This report applies the high-resolution melt (HRM) feature of RT-qPCR to detect shifts caused by the presence of gold nanoparticles (AuNPs). A universal RNA standard (untreated control) sample was spiked with known amounts of AuNPs and reverse transcribed, where 10 reference genes were amplified. The amplification plots, dissociation assay (melt) profiles, electrophoretic profiles and HRM difference curves were analysed and detected interference caused by AuNPs, which differed according to the amount of AuNP present (i.e. semi-quantitative). Whether or not the assay interference was specific to the reverse transcription or the PCR amplification step was tested. The study was extended to a target gene-of-interest (GOI), Caspase 7. Also, the effect on in vitro cellular samples was assessed (for reference genes and Caspase 7). This method can screen for the presence of AuNPs in RNA samples, which were isolated from biological material in contact with the nanomaterials, i.e., during exposure and risk assessment studies. This is an important quality control procedure to be implemented when quantifying the expression of a GOI from samples that have been in contact with various ENMs. It is recommended to further examine 18S, PPIA and TBP since these were the most reliable for detecting shifts in the difference curves, irrespective of the source of the RNA, or, the point at which the different AuNPs interacted with the assay.

High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides.

Current real-time high-throughput Polymerase Chain Reaction (qPCR) methods do not distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009-2011), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92-101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/mL of sample. There was high concordance (Kappa = 0.73-0.92); sensitivity (85-100%) and specificity (96-100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

Selection and validation of reference genes for normalization of qRT-PCR data to study the cannabinoid pathway genes in industrial hemp.

There has been significant interest in researching the pharmaceutical applications of Industrial hemp since its legalization three years ago. The crop is mostly dioecious and known for its production of phytocannabinoids, flavonoids, and terpenes. Although many scientific reports have showed gene expression analysis of hemp through OMICs approaches, unreliable reference genes for normalization of qRT-PCR data make it difficult to validate the OMICs data. Four software packages: geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the differential gene expression patterns of 13 candidate reference genes under osmotic, heavy metal, hormonal, and UV stresses. EF-1α ranked as the most stable reference gene across all stresses, TUB was the most stable under osmotic stress, and TATA was the most stable under both heavy metal stress and hormonal stimuli. The expression patterns of two cannabinoid pathway genes, AAE1 and CBDAS, were used to validate the reliability of the selected reference genes. This work provides useful information for gene expression characterization in hemp and future research in the synthesis, transport, and accumulation of secondary metabolites.

Identification of the best housekeeping gene for RT-qPCR analysis of human pancreatic organoids.

In the last few years, there has been a considerable increase in the use of organoids, which is a new three-dimensional culture technology applied in scientific research. The main reasons for their extensive use are their plasticity and multiple applications, including in regenerative medicine and the screening of new drugs. The aim of this study was to better understand these structures by focusing on the choice of the best housekeeping gene (HKG) to perform accurate molecular analysis on such a heterogeneous system. This feature should not be underestimated because the inappropriate use of a HKG can lead to misleading data and incorrect results, especially when the subject of the study is innovative and not totally explored like organoids. We focused our attention on the newly described human pancreatic organoids (hPOs) and compared 12 well-known HKGs (ACTB, B2M, EF1α, GAPDH, GUSB, HPRT, PPIA, RNA18S, RPL13A TBP, UBC and YWHAZ). Four different statistical algorithms (NormFinder, geNorm, BestKeeper and ΔCt) were applied to estimate the expression stability of each HKG, and RefFinder was used to identify the most suitable genes for RT-qPCR data normalization. Our results showed that the intragroup and intergroup comparisons could influence the best choice of the HKG, making clear that the identification of a stable reference gene for accurate and reproducible RT-qPCR data normalization remains a critical issue. In summary, this is the first report on HKGs in human organoids, and this work provides a strong basis to pave the way for further gene analysis in hPOs.

A method for measuring the experimental resolution of laboratory assays (clinical biochemical, blood count, immunological, and qPCR) to evaluate analytical performance.

BACKGROUND: The measurement method for experimental resolution and related data to evaluate analytical performance is poorly explored in clinical research. We established a method to measure the experimental resolution of clinical tests, including biochemical tests, automatic hematology analyzer methods, immunoassays, chemical experiments, and qPCR, to evaluate their analytical performance. METHODS: Serially diluted samples in equal proportions were measured, and correlation analysis was performed between the relative concentration and the measured value. Results were accepted for p ≤ 0.01 of the correlation coefficient. The minimum concentration gradient (eg, 10%) was defined as the experimental resolution. For this method, the smaller the value, the higher the experimental resolution and the better the analytical performance. RESULTS: The experimental resolution of the most common biochemical indices reached 10%, with some even reaching 1%. The results of most counting experiments showed experimental resolution up to 10%, whereas the experimental resolution of the classical chemical assays reached 1%. Unexpectedly, the experimental resolution of more sensitive assays, such as immunoassays was only 25% when using the manual method and 10% for qPCR. CONCLUSION: This study established a method for measuring the experimental resolution of laboratory assays and provides a new index for evaluating the reliability of methods in clinical laboratories.

Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.