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Evaluation of inactivated snakehead rhabdovirus as an internal positive control for rt-qpcr diagnosis of viral hemorrhagic septicemia virus in fish

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JOURNAL OF VIROLOGICAL METHODS 2025年 334卷 115128页

作者： Choi, Myoung Gwang Kim, Do-Hyung Kim, Hyoung Jun Kim, Ki Hong Pukyong Natl Univ Dept Aquat Life Med Busan 48513 South Korea Natl Inst Fisheries Sci WOAH Reference Lab VHS Busan 46083 South Korea

Viral hemorrhagic septicemia virus (VHSV) is a significant pathogen causing mass mortalities in marine and freshwater fish worldwide. Accurate diagnosis through quantitative reverse transcription PCR (rt-qpcr) is essential to prevent its spread, but false negatives can compromise results. In this study, we evaluate the use of heat-inactivated snakehead rhabdovirus (SHRV) as an internal positive control (IPC) for VHSV diagnosis. SHRV's similarity to VHSV in viral structure and genome makes it an ideal IPC. The introduction of SHRV IPC into the rt-qpcr workflow can improve the reliability of diagnostic results by enabling the detection of technical failures during the RNA extraction or amplification process. Our results show that heat-inactivated SHRV preserved RNA integrity for IPC use, and SHRV IPC can provide a useful tool for detecting and interpreting false negatives without affecting assay sensitivity.

关键词： VHSV diagnosis rt-qpcr False negatives SHRV Internal positive control

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Development and evaluation of a SYBR Green-based rt-qpcr assay with a specific primer set for tomato seed testing against tomato brown rugose fruit virus

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JOURNAL OF GENERAL PLANT PATHOLOGY 2025年第3期91卷 160-170页

作者： Ota, Emi Shinosaka, Hibiki Ishibashi, Kazuhiro Takeyama, Sawana Matsuyama, Momoko Tomitaka, Yasuhiro Matsushita, Yosuke Osaki, Kohei Kubota, Kenji NARO Inst Plant Protect Div Core Technol Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan NARO Ctr Seeds & Seedlings Dept DUS Test & Seed Inspect 2-2 Fujimoto Tsukuba Ibaraki 3050852 Japan NARO Inst Agrobiol Sci Div Plant Mol Regulat Res 2-1-2 Kannondai Tsukuba Ibaraki 3058602 Japan NARO Inst Plant Protect Div Crop Pest Control Res 2-1-18 Kannondai Tsukuba Ibaraki 3058666 Japan

Since its first occurrence in Israel and Jordan in 2014 and 2015, the tomato brown rugose fruit virus (ToBRFV) has become one of the most concerning pathogens affecting tomatoes and other crops worldwide. Its rapid spread is believed to result from the international trade of contaminated seeds and its seed transmissibility, underscoring the critical importance of seed health testing for ToBRFV to prevent further dissemination of the virus. To this end, reverse transcription-quantitative polymerase chain reaction (rt-qpcr) protocols employing TaqMan probe chemistry have been widely adopted. However, the development of rt-qpcr protocols for ToBRFV seed testing using SYBR Green chemistry remains limited. The SYBR Green method offers the advantage of distinguishing ToBRFV from other tobamoviruses through melt curve analysis. In this study, we developed a SYBR Green-based rt-qpcr detection method using newly designed primer sets, which demonstrated high specificity for ToBRFV and sufficient sensitivity. While this protocol requires further optimization and validation for application in routine seed testing, it establishes a foundational approach for SYBR Green-based rt-qpcr seed testing. Additionally, this study raises an important question regarding the relationship between rt-qpcr results in seed tests and the likelihood of virus contamination or transmission via seeds.

关键词： ToBRFV Tobamoviruses SYBR green rt-qpcr Melt curve Tomato

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Comparison of rt-qpcr With Branched DNA to Quantify a Lipid Nanoparticle-Encapsulated mRNA Therapeutic in Serum and Liver Tissue Samples From Nonclinical PK Studies

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AAPS JOURNAL 2025年第1期27卷 1-10页

作者： Ortiz, Jessica Brunner, Laura Ci, Lei Baek, Rena Jani, Darshana Marshall, Jean-Claude Pennucci, Jason Moderna Inc Cambridge MA 02142 USA

While the branched DNA (bDNA) assay is an established bioanalytical method for measurement of lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) pharmacokinetic parameters, reverse transcription-quantitative polymerase chain reaction (rt-qpcr) has been considered as an alternative platform. rt-qpcr and bDNA platforms were compared for sensitivity, specificity, correlation, and overall assay performance using serum and tissue samples from 2 nonclinical mouse studies of a therapeutic mRNA candidate, LNP-PAH-mRNA, which encodes for human phenylalanine hydroxylase enzyme. Pharmacokinetic parameter noncompartmental analysis was completed using Phoenix WinNonlin. The assays were compared using simple linear regression and Bland-Altman analyses. Sensitivity ranged from 0.05 to 6.40 ng/mL for the bDNA assays, from 0.00000761 to 7.61 ng/mL in serum, and from 0.000179 to 179 ng/g in tissue for the rt-qpcr assay. Inter-assay accuracy was within +/- 10%, inter-assay precision was <= 10%, and the total error for both assays was <= 20%. rt-qpcr serum mRNA concentrations were 2- to fourfold lower compared with the bDNA assay, whereas tissue samples were comparable between assays. A linear relationship with - 0.37 to - 0.02 systematic bias demonstrated acceptable concordance. Bland-Altman plots demonstrated close equivalence, with a negative bias of < 0.5, and >= 95% of the data points were within the 95% limits of agreement. The comparison of the rt-qpcr with bDNA assay platforms for quantification of pharmacokinetic properties of an mRNA-LNP therapeutic has demonstrated acceptable concordance. This comparison reinforces the use of the rt-qpcr, a widely accessible strategy, as an alternative platform for the quantification of subsequent mRNA-LNP therapeutics.

关键词： bDNA bioanalytical mRNA therapeutics pharmacokinetics rt-qpcr

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Detection of Porcine Norovirus GII.18 Strains in Pigs Using Broadly Reactive rt-qpcr Assay for Genogroup II Noroviruses

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FOOD AND ENVIRONMENTAL VIROLOGY 2025年第1期17卷 1-8页

作者： Gupta, Ankita K. Heinonen, Mari Konig, Emilia Mikkonen, Venla Maunula, Leena Univ Helsinki Fac Vet Med Dept Food Hyg & Environm Hlth Helsinki Finland Univ Helsinki Fac Vet Med Dept Prod Anim Med Helsinki Finland

Noroviruses, belonging to the family Caliciviridae, are classified into at least ten genogroups (G) based on their major capsid protein (VP1). The common genogroup to be identified in both humans and pigs is GII, although porcine noroviruses (PoNoVs) belong to genotypes of their own (GII.11, GII.18, and GII.19). So far, PoNoVs have not been studied much in Finland, possibly due to their rather symptomless nature in pigs. In the present study, we enrolled a total of 189 fecal samples collected from pigs from Finnish farms. Samples were taken from 12 farms in 2010, 2019 and 2020. We analyzed feces from growing pigs ranging from 2.1 to 6 months of age. RNA was extracted from fecal suspensions using a commercial viral RNA extraction kit, followed by rt (reverse transcription)-qpcr. The genotypes were determined by Sanger sequencing of the PCR fragments amplified by conventional PCR. Of the 12 farms, 6 (50%) had at least one PoNoV-infected pig. Altogether 18 (9.5%) of the 189 pigs tested positive for PoNoVs. Pigs mostly aged over 4 months were infected with PoNoVs. Eventually, 12 positive samples were determined as genotype GII.18. We could demonstrate the presence of PoNoVs in Finnish pigs. In future, more studies in which longer sequences from PoNoV genome can be obtained, are required.

关键词： Caliciviridae Human norovirus Porcine norovirus rt-qpcr Genotyping

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Evaluation of suitable reference genes for gene expression studies in the developing mouse cortex using rt-qpcr

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BMC NEUROSCIENCE 2025年第1期26卷 1-11页

作者： Uppalapati, Ananya Wang, Timothy Nguyen, Lena H. Univ Texas Dallas Sch Behav & Brain Sci Dept Neurosci Richardson TX 75080 USA

BackgroundReal-time quantitative PCR (rt-qpcr) is a widely used method to investigate gene expression in neuroscience studies. Accurate relative quantification of rt-qpcr requires the selection of reference genes that are stably expressed across the experimental conditions and tissues of interest. While rt-qpcr is often performed to investigate gene expression changes during neurodevelopment, few studies have examined the expression stability of commonly used reference genes in the developing mouse ***, we evaluated the stability of five housekeeping genes, Actb, Gapdh, B2m, Rpl13a, and Hprt, in cortical tissue from mice at embryonic day 15 to postnatal day 0 to identify optimal reference genes with stable expression during late corticogenesis. The expression stability was assessed using five computational algorithms: BestKeeper, geNorm, NormFinder, DeltaCt, and RefFinder. Our results showed that B2m, Gapdh, and Hprt, or a combination of B2m/Gapdh and B2m/Hprt, were the most stably expressed genes or gene pairs. In contrast, Actb and Rpl13a were the least stably *** study identifies B2m, Gapdh, and Hprt as suitable reference genes for relative quantification in rt-qpcr-based cortical development studies spanning the period of embryonic day 15 to postnatal day 0.

关键词： Cerebral cortex Neurodevelopment Mouse rt-qpcr Gene expression Reference genes Housekeeping genes

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Design, development, and validation of a strand-specific rt-qpcr assay to differentiate replicating versus nonreplicating Rabies virus

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JOURNAL OF VIROLOGICAL METHODS 2025年 332卷 115054页

作者： Xiang, Hong Hong, Chao Tian, Yuan Gao, Yu Cun, Yina Zhao, Hongling Li, Guilan Chen, Yu Zhou, Jian Chinese Acad Med Sci & Peking Union Med Coll Inst Med Biol Kunming Peoples R China

The Rabies virus, a single-strand RNA virus with a negative-sense polarity, is responsible for causing encephalitis and is a zoonotic disease. If not promptly treated after infection, it has a close to 100 % fatality rate. Similar to other negative-sense polarity single-strand RNA viruses, the Rabies virus requires the creation of a positive-strand RNA intermediate for replication. One approach to identify this replication activity is to detect the complementary strand of the viral RNA genome in suspected infected cells or tissues. The reported Rabies virus rt-qpcr detection methods are designed to detect total viral load in samples without distinguishing between positive- and negative-strand for RNA viruses. As such, in this study, a sensitive Taqman-based strand-specific rt-qpcr assay has been developed to quantitatively detect both the positive- and negative-strand of the Rabies virus. This method demonstrates good reproducibility across a wide dynamic range and exhibits linearity of 8 logs with a lower limit of detection of 103 copies/mu L for the positive-strand and 9 logs with a lower limit of detection of 102 copies/mu L for the negative-strand. Notably, it can accurately detect a specific viral RNA strand even in the presence of high levels of the opposite strand, confirming the method's specificity. In summary, a reliable strandspecific rt-qpcr assay has been developed and validated to differentiate replicating from non-replicating Rabies virus.

关键词： Rabies virus rt-qpcr Strand-specific rt-qpcr Single-strand RNA virus Virus detection

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MB based rt-qpcr increase the clinical application of cfEBV DNA for NPC in non-endemic area of China

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SCIENTIFIC REPOrtS 2025年第1期15卷 1-12页

作者： He, Qiao Tuo, Yi Zhou, Yi Yan, Yue Liu, Xin Zhao, Dan Wang, Qiuju Luo, Hao Zhang, Zhengyao Meng, Fanping Ying, Binwu Wang, Dongsheng Yang, Mu Huang, Yecai Univ Elect Sci & Technol China Sichuan Canc Hosp & Inst Sichuan Clin Res Ctr Canc Sichuan Canc CtrDept Clin LabSch Med Chengdu 610000 Peoples R China Sichuan Univ West China Hosp Dept Lab Med Chengdu 610041 Peoples R China Chengdu Univ Tradit Chinese Med Coll Med Technol Chengdu 611137 Peoples R China Chong Qing Univ Dept Clin Lab Three Gorges Hosp Chongqing Peoples R China Univ Elect Sci & Technol China Sichuan Canc Hosp & Inst Sichuan Clin Res Ctr Canc Sichuan Canc CtrDept Expt Res Chengdu 610000 Peoples R China Univ Elect Sci & Technol China Dept Radiat Oncol Radiat Oncol Key Lab Sichuan Prov Sichuan Clin Res Ctr CancSichuan Canc Hosp & Inst Chengdu 610000 Peoples R China Univ Elect Sci & Technol China Sichuan Canc Hosp & Inst Sichuan Canc Ctr Dept Clin LabSichuan Clin Res Ctr Canc Chengdu 610000 Sichuan Peoples R China

To compare the performance of magnetic bead (MB) and the concentrated precipitation (CP) based rt-qpcr to qualify cell free EBV DNA (cfEBV DNA) for nasopharyngeal carcinoma (NPC) in non-endemic area of China. From January 2014 to June 2024, a retrospective analysis of 2 cohort studies on cfEBV DNA in NPC patients was conducted to assess the diagnostic value, positive detection rate and clinical application. cfEBV DNA detection with CP based rt-qpcr in cohort 1 and MB based rt-qpcr method in cohort 2. The MB based rt-qpcr for the quantitative measurement of cfEBV DNA load was higher than the CP based rt-qpcr in the same plasma samples from NPC patients (P < 0.001). CP based rt-qpcr measured cfEBV DNA in 1405 NPC and 244 healthy controls in cohort 1 with 40.8% sensitivity (AUC = 0.704, 95% CI: 0.676-0.731). In cohort 2(683 naive NPC and 303 controls), cfEBV DNA had a sensitivity of 75.84% (AUC = 0.879, 95% CI: 0.86-0.90). There were no significant differences in TNM stage among NPC between the two cohorts (P > 0.05). The MB method considerably increased the positive detection rate of cfEBV DNA in NPCs at stages III-IV, T2-T4, N1-N3, and M0 (P < 0.001). At the end of treatment, 97.51% of patients had no detectable EBV and just 2.49% had detectable cfEBV DNA. Those who received <= 2 or >= 3 cycles of NAC had a median t(1/2) clearance rate of 9.8 days and 12.6 days, respectively. MB based rt-qpcr increased the quantity of cfEBV DNA. MB based rt-qpcr demonstrated superior sensitivity and positive detection rates for cfEBV DNA. cfEBV DNA can be more positively noticed, with a higher diagnostic value and a broader variety of clinical applications among NPC in non-endemic areas.

关键词： rt-qpcr MB CP cfEBV DNA NPC

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Integrating Raman spectroscopy and rt-qpcr for enhanced diagnosis of thyroid lesions: A comparative study of biochemical and molecular markers

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JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS 2025年 261卷 116844页

作者： Neto, Lazaro P. M. dos Santos, Laurita Carvalho, Luis Felipe C. S. Santos, Andre B. O. Martin, Airton A. Canevari, Renata A. Univ Vale Paraiba UNIVAP Inst Pesquisa & Desenvolvimento Ave Shishima Hifumi 2911 BR-12244000 Sao Jose Dos Campos SP Brazil Pontificia Univ Catolica PUC Minas Dept Ciencias Biol & Saude Ave Padre Cletus Francis Cox 1661 BR-37714620 Pocos De Caldas MG Brazil Univ Brasil Inst Pesquisa Campus Sao PauloRua Carolina Fonseca 235 BR-08230030 Sao Paulo SP Brazil Univ Taubate UNITAU Programa Posgrad Ciencias Saude Taubate Brazil Univ Sao Paulo Hosp Clin Fac Med Ave Doutor Arnaldo 455 BR-01246903 Sao Paulo SP Brazil

Thyroid cancer is the most prevalent endocrine malignancy, with increasing incidence due to advancements in diagnostic techniques. Ultrasound (US) and fine needle aspiration (FNA) cytology, widely used in clinical practice, have detection accuracies ranging from 65 % to 95 %. However, these methods may yield inconclusive or difficult-to-interpret results, emphasizing the need for complementary diagnostic techniques. This study explores the integration of Raman spectroscopy and gene expression analysis via rt-qpcr to improve the diagnosis of thyroid lesions, classified into groups: follicular thyroid carcinoma (FTC), papillary thyroid carcinoma (PTC) and goiter tissues. Healthy tissue samples were used as normalizing controls in both analysis. Raman spectroscopy analyzed 35 samples, while rt-qpcr assessed 33 samples. For comparison, the same 19 samples previously analyzed by both techniques were examined. Raman spectroscopy, a non-invasive technique, has shown effectiveness in distinguishing between benign and malignant thyroid tissues by identifying key biochemical components such as DNA, RNA, proteins, and lipids. The distinguishing of FTC from goiter using Raman spectroscopy achieved an accuracy rate of 82.3 %. Gene expression analysis via rt-qpcr focused on six genes: TG, TPO, PDGFB, SERPINA1, TFF3, and LGALS3. Specifically, SERPINA1 was overexpressed in PTC, TFF3 showed elevated levels in FTC, and LGALS3 was elevated in both PTC and FTC compared to goiter and normal tissues. These findings align with existing literature, suggesting that these genes could serve as valuable diagnostic molecular markers. The expression analysis of these genes within this subset of samples demonstrated concordance with the classification derived from PCA of Raman spectroscopy data. The integration of Raman spectroscopy and rtqpcr offers a complementary approach to traditional histological analysis, providing enhanced sensitivity and specificity in diagnosing thyroid lesions.

关键词： Thyroid Diagnosis Raman spectroscopy rt-qpcr

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Diagnosis of SARS-CoV-2: Experience with rapid immunochromatography tests and rt-qpcr

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JOURNAL OF CLINICAL VIROLOGY PLUS 2025年第2期5卷 100207-100207页

作者： Gaytan, David Alejandro Cabrera Bautista, Porfirio Felipe Hernandez Tinoco, Clara Esperanza Santacruz Yaah, Julio Elias Alvarado Hernandez, Yu-Mei Anguiano Miguel, Bernardo Martinez Paras, Alfonso Vallejos Nieto, Lumumba Arriaga Gutierrez, Nancy Sandoval Muniz, Concepcion Grajales Paz, Alejandro Moctezuma Betancourt, Leticia Jaimes Inst Mexicano Seguro Social Coordinac Calidad Insumos & Labs Especializados Ciudad De Mexico Mexico Inst Mexicano Seguro Social Coordinac Vigilancia Epidemiol Ciudad De Mexico Mexico Inst Mexicano Seguro Social Bienestar IMSS BIENEST Organismo Publ Descentralizado Serv Salud Ciudad De Mexico Mexico Inst Mexicano Seguro Social Coordinacio Invest Salud Ciudad De Mexico Mexico Inst Mexicano Seguro Social Unidad Med Familiar 7 Ciudad De Mexico Mexico

Background: Mexico has experienced six waves were experiences between 2019 and 2023 of coronavirus disease 2019 (COVID-19). In the first wave, diagnosis relied on rt-qpcr;rapid tests for SARS-CoV-2 were introduced from the second wave onward. However, the patterns of results with this test in Mexico have not been documented in population-based studies. Objective: To describe the temporal patterns of the results from rt-qpcr and rapid immunochromatography tests for SARS-CoV-2 detection from the second to the sixth epidemic wave in Mexico. Materials and methods: This was a multicenter study at the national level. The operational definition of suspected cases of viral respiratory illness, confirmed cases of SARS-CoV-2 in Mexico, and data for asymptomatic individuals who required rapid testing between 2020 and 2023 were employed;testing was employed for the entirety of the four years. Positivity was estimated per epidemiological week, state, and condition for both tests. Spearman's correlation coefficient, trend chi-square analysis, and odds ratios with 95 % confidence intervals were used. Results: A total of 1749,765 cases had rt-qpcr data recorded from 2020 to 2023, and 8356,903 cases with rapid tests were conducted for virus identification. Compared with the rest of the country, the southeastern region exhibited different patterns. The positivity rate of rapid tests from the epidemiological surveillance system was lower than that of rt-qpcr during interepidemics periods, whereas the positivity rate of rapid tests for symptomatic individuals was higher than that of rt-qpcr tests over two years. Conclusion: Rapid tests for SARS-CoV-2 identification in Mexico were affordable and timely at the local level. These tests revealed differing epidemic wave patterns in the southeastern region of the country.

关键词： rt-qpcr SARS-CoV-2 Rapid antigen test Epidemiological surveillance RdRp gene

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High-throughput sperm screening using one-step rt-qpcr: Improvement and re-evaluation

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ANALYTICAL BIOCHEMISTRY 2025年 698卷 115727页

作者： Kubo, Seiji Amai, Keito Nakano, Fumitaka Tanaka, Jin Niimi, Hideki Univ Toyama Fac Med Dept Clin Lab & Mol Pathol Acad Assembly 2630 Sugitani Toyama 9300194 Japan Ishikawa Prefectural Police Headquarters Forens Sci Lab 1-1 Kuratsuki Kanazawa 9208553 Japan

Sperm identification is crucial in sexual assault cases. While microscopic analysis is the gold standard for sperm detection, it is a laborious procedure even for trained personnel. Reverse transcription-quantitative real-time PCR (rt-qpcr) can enhance the screening by detecting sperm-specific mRNA markers, such as protamine 2 (PRM2). This study aimed to develop a one-step rt-qpcr assay targeting PRM2 mRNA. Our assay was capable of detecting as low as 0.01 mu L of semen with high specificity and demonstrated successful detection of PRM2 mRNA in simulated-case samples. Owing to the simple workflow involved, our assay requires <30 min for RNA extraction and <60 min for rt-qpcr. Our assay enables high-throughput sperm screening and offers a promising strategy for enhancing the workflow of sexual assault cases.

关键词： Semen Sperm PRM2 rt-qpcr

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