TaqMan Real-time Quantitative PCR Research Articles

Real-time quantitative reverse transcription PCR assay for the detection of Nuomin virus – An emerging tick-borne virus

Nuomin virus (NOMV), an emerging tick-borne virus (TBVs) identified in 2020, has been associated with fever, headache, and potential liver dysfunction in infected individuals. This study presents a novel TaqMan real-time quantitative PCR method designed for the rapid, sensitive, and specific detection of NOMV, facilitating early diagnosis. Utilizing Beacon Designer software 8.0, we optimized the PCR assay including the development of primers and probes to precisely target the conserved region of the NOMV genome, followed by optimization of primer and probe concentrations and annealing temperature. The resulting assay demonstrated robust performance, with standard curve represented by the equation y=−3.29x+39.42, a high correlation coefficient (R2 = 0.995) and an efficiency 99.53 %. Importantly, the method exhibited exceptional specificity, which did not yield cross-reacting signals from other TBVs, including Songling virus (SGLV), Beiji virus (BJNV), tick-borne encephalitis virus (TBEV), Yezo virus (YEZV), Alongshan virus (ALSV), and severe fever with thrombocytopenia syndrome bunyavirus (SFTSV). The assay’s detection limit was remarkably low, reaching 10 copies/μL, representing a 100-fold increase compared to semi-nested RT-PCR. Additionally, it demonstrated excellent repeatability, with coefficients of variation for intra- and inter-group tests consistently below 3 %. Clinical evaluations confirmed the assay’s superior performance, highlighting its high specificity, sensitivity, and reproducibility for NOMV detection. In conclusion, the method developed in this study provides a valuable tool to support timely management of NOMV infections, with significant implications for clinical practice.

Dynamic of Puccinia striiformis f. sp. tritici urediniospore and its correlation with wheat stripe rust epidemics in Xiangyang of China

Stripe rust is one of the main diseases of wheat, which seriously threatens wheat production and food security all over the world. Xiangyang located in the Northwest of Hubei province in China is one of the main winter propagation and spring epidemic regions of Puccinia striiformis f. sp. tritici (Pst), which can provide urediniospores to the major wheat-growing regions in eastern and northeastern China. Understanding the dynamic of Pst urediniospore is important for giving prediction of wheat stripe rust epidemic for eastern and northeastern China and controlling the epidemic of wheat stripe rust. In this study, spore trapper and TaqMan real-time quantitative PCR (TaqMan-qPCR) detection system were employed to monitor Pst urediniospore from December 2018 to December 2022 in Xiangyang. Weather variables including air temperature, relative humidity, sunshine duration and rainfall were collected to clarify the relationship with urediniospore density in the air. In addition, the relationship between disease index of wheat stripe rust and urediniospore density in the air was analyzed. Results showed that Pst urediniospore could be captured in the air all year round. The order of the density of urediniospore from most to least was from April to June, October to December, January to March, and July to September except 2022. The urediniospore density reached the peak when the air temperature was 10–22 °C and the relative humidity was 70%∼85% from April to May in 2019, 2020 and 2021. The density of Pst urediniospores from February to April was linearly related to the total precipitation of 25 days prior to the final day of a 7-day trapping period. There was a significant positive correlation between the disease index of wheat stripe rust and the cumulative urediniospore density 2–4 weeks before the investigation date of wheat stripe rust from March to May (P < 0.05). There was no significant correlation between the disease index and the cumulative urediniospore density from 1–4 weeks after the investigation date of stripe rust from March to May (P > 0.05). This study laid a foundation for the establishment of wheat stripe rust prediction model based on urediniospore density and meteorological factors.

Load of the ash dieback pathogen hymenoscyphus fraxineus differs in soil

AbstractThe ascomycete Hymenoscyphus fraxineus causes the devastating ash dieback disease of European ash (Fraxinus excelsior L.). Spore traps are often used to measure the amount of ascospores in the environment, but the pathogen-load of the soil in ash stands has not been recorded so far. This is of particular interest with regard to the occurrence of ash stem necrosis, a decisive factor for the severe course of the disease. In order to gain a more differentiated insight into the pathogen-load in ash stands, we analysed soil samples from four ash tree sites in southern Germany, covering a clone plantation, two seed orchards and a forest. The pathogen-load was determined using a quantitative TaqMan real-time PCR assay for ten to twenty plots per stand. Results obtained by the species-specific assay highlighted that the pathogen-load is heterogeneously distributed in the ash stands. H. fraxineus DNA targets were detected in 17% of the soil samples. The pathogen-load differed according to soil depth, with the highest pathogen abundance in the top 5 cm, followed by 5–10 cm and finally 10–15 cm. Pathogen-load and thereby infection pressure were found to be highly variable for the individual trees in one stand. Overall, the study discovered detectable levels of H. fraxineus in the soil of all four study sites, which supports the hypothesis that H. fraxineus can be found in the soil of ash stands. The qPCR approach was found to be an effective method for monitoring the load of H. fraxineus in soil and for demonstrating the successful application of the method on the sample type of custom-made spore traps. Results suggest the implication of site-specific pathogen-load determination in future H. fraxineus-monitoring and selection of less susceptible ash trees for breeding and seed production.

Dynamics of Puccinia striiformis f. sp. tritici Urediniospores and Its Relationship with Meteorological Factors in Mianyang, China.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the main diseases of wheat worldwide. Mianyang of Sichuan province in Southwest China is one of main regions for winter Pst inoculum production and spring epidemic and provides urediniospores for infecting wheat in the surrounding regions. Understanding the urediniospore dynamics is important to predict and manage stripe rust. In this study, spore trapping coupled with a TaqMan real-time quantitative PCR method was used to monitor airborne Pst urediniospores from December 2019 to December 2022 in Mianyang. Weather conditions (temperature, relative humidity, daily sunshine duration, and precipitation) were collected for the same period. These data were used to study the relationship of airborne urediniospore density with climatic conditions. The results showed that Pst urediniospores were captured all year round, and the annual peak of urediniospore densities occurred in the period from March to April in which the urediniospores accounted for the largest proportion of the annual total urediniospores. The density of urediniospores in the period of March to April was linearly related to the average sunshine duration of 20 days and average temperature of 15 days prior to the final day of a 7-day trapping period. This relationship needs to be tested in other regions where Pst can sporulate during the winter before it can be integrated with Pst infection conditions to predict rust development.

TaqMan real-time quantitative PCR for the detection of beef tallow to assess the authenticity of edible oils

TaqMan real-time quantitative PCR for the detection of beef tallow to assess the authenticity of edible oils

Development of a highly sensitive TaqMan method based on multi-probe strategy: its application in ASFV detection.

The establishment of high sensitive detection method for various pathogenic microorganisms remains constantly concerned. In the present study, multi-probe strategy was first systematically investigated followed by establishing a highly sensitive TaqMan real-time fluorescent quantitative PCR (qPCR) method for detecting African swine fever virus (ASFV). Briefly, four probes based on the B646L gene of ASFV were designed and the effects of different combinations of the probes in a single TaqMan qPCR assay on the detection sensitivity were investigated. As less as 0.5-5 copies/μl of the ASFV gene was detected by the established TaqMan qPCR assay. Furthermore, plasmid harboring the B646L in water samples could be concentrated 1000 times by ultrafiltration to enable a highly sensitive detection of trace viral nucleic acids. Moreover, no cross-reactivity was observed with other common clinical swine viruses such as PCV2, PCV3, PCV4, PEDV, PDCoV, CSFV, PRRSV, and PRV. When detecting 173 clinical porcine serum samples, the coincidence rate between the developed method and WOAH (World Organization of Animal Health) recommended method was 100%. This study might provide an integrated strategy to achieve higher detection sensitivity of trace pathogenic microorganisms and applicably sensitive TaqMan-based qPCR assays.

Inhibitory effect of Streptococcus mutans antisense vicK RNA regulating the cariogenicity of oral streptococci multi-species biofilm

Objective: To investigate the regulative effects of Streptococcus mutans (Sm) antisense vicK RNA (ASvicK) on the multi-species biofilm formed by three common oral streptococci (Sm, Streptococcus sanguinis, and Streptococcus gordonii) (Sm+Ss+Sg). Methods: ASvicK over-expression strain was constructed by using a recombinant plasmid, and three-species biofilm UA159+Ss+Sg and ASvicK+Ss+Sg were cultured. The phenotypes of biofilms were detected by scanning electron microscopy (SEM). Crystal violet (CV) assay was used to detect biofilm biomass. Lactate kit and anthrone-sulfuric acid colorimetric assay were used to determine the abilities of lactic acid and exopolysaccharides production, respectively. The proportions of three-species and expression levels of the cariogenic-related genes in biofilms were detected by TaqMan fluorescence quantitative PCR and real-time fluorescence quantitative PCR. A biofilm demineralization model of human enamel slabs was further constructed, and the hardness of enamel surface was detected. Results: Compared to UA159+Ss+Sg, over-expression of ASvicK could inhibit biofilm formation and lactic acid production in ASvicK+Ss+Sg biofilm significantly decreased by 78.93% (P<0.001) and 62.23% (P<0.001), respectively. With ASvicK over-expression, the amounts of water-insoluble and-soluble glucoses in ASvicK+Ss+Sg biofilm were reduced respectively by 39.13% (P<0.001) and 68.00% (P<0.001). Compared to the UA159+Ss+Sg Group, the proportion of Sm, the cariogenic bacteria, showed 33.00% reduction (P<0.01) in Sm+Ss+Sg biofilm, and the gene expressions of cariogenic-relative genes vicK/X, gtfB/C/D, and ftf significantly decreased (P<0.05). The micro-hardness value of enamel slabs after demineralization by ASvicK+Ss+Sg biofilm was significantly increased to 183.84% (P<0.001). Conclusions: ASvicK over-expression could reduce the Sm proportion and weaken the cariogenicity of oral Streptococcus biofilm, thereby possibly slowing down the progression of caries.

Optimization and Application of Quantitative In-House ELISA for Diagnosis of HBsAg and its Correlation with Commercial ELISA and Molecular Kits

Background: Several studies have indicated the role of quantitative hepatitis B surface antigen (HBsAg) evaluation in managing and prognosis of hepatitis B virus (HBV) infection. Thus, quantitative evaluation of HBsAg using cost-effective assays can be an important approach to managing HBV patients. Objectives: This study aimed to set up and apply an in-house quantitative enzyme-linked immunosorbent assay (ELISA) to evaluate HBsAg in research and diagnosis. Methods: New Zealand white rabbits were immunized with HBsAg. Sera were collected 28 days after immunization, and polyclonal HBsAb (antibody to hepatitis B surface antigen) were purified and evaluated. Then, the in-house quantitative ELISA was optimized. Finally, the functional characteristics of the assay were evaluated using 200 plasma samples compared to commercial ELISA and quantitative TaqMan real-time PCR. Results: The assay has a limit of detection (LOD) of 0.5 ng/mL with a specificity and sensitivity of 94% and 97%, respectively. The assay's highest coefficient of variation (CV) values for intra- and inter-assays were 7.23% and 8.59%, respectively. The correlation of the developed assay with commercial ELISA was 0.987 (P-value = 0.024). The correlations of the developed assay and commercial ELISA with quantitative TaqMan real-time PCR were 0.739 and 0.658, respectively (P-value = 0.017). Conclusions: The developed assay has a suitable sensitivity and specificity. It is also reproducible and well-correlated with commercial assays. Most importantly, it is cost-effective and, thus, can be used for detecting and quantifying HBsAg in research and diagnosis.

Dynamic of Puccinia striiformis f. sp. tritici Urediniospore in Longnan, a Critical Oversummering Region of China.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici Erikss (Pst), is a devastating disease resulting in yield reduction. Because the temperature limits the overwintering and oversummering of Pst, and it cannot complete the whole year cycle in most areas of China. Longnan located in the southeast of Gansu province is one of the annual cycle areas of Pst, which can supply urediniospores in autumn to eastern wheat-growing areas in China. In this study, a TaqMan real-time quantitative PCR (TaqMan-qPCR) detection system for Pst urediniospores was established, and the detection limit was a single urediniospore. The dynamics of Pst urediniospores in Longnan were monitored by spore trapping and TaqMan-qPCR for 3 years. Meanwhile, the meteorological conditions including air temperature, relative humidity and precipitation were recorded. Results showed that Pst urediniospores can be captured from March to December, and two peaks of urediniospore number appeared in May and June, respectively. The number of urediniospore is closely related to temperature and precipitation from March to June. In addition, we found that the number of Pst urediniospore had the peak value when the average air temperature was 10~21℃ and the relative humidity was 60%~85% from May to June. The exponential model could describe the variation of Pst urediniospore number based on average temperature and precipitation from March to June. It is worth mentioning that the exponential model based on average temperature 7 days before spore capture has a great advantage in predicting the urediniospore density in the air. This study laid a foundation for establishment of a prediction model for wheat stripe rust based on the number of urediniospores and meteorological factors.

Impaired CD4+ Cytotoxic T Lymphocyte Activity in Polyomavirus BK Infected Kidney Transplant Recipients.

The reactivation of polyomavirus BK (BKPyV) contributes to increased morbidity and mortality rates of transplant patients, especially kidney transplant recipients (KTRs). CD4+ T cells are important immune cells active during BKPyV infection in KTRs. This research tried to examine the phenotype of CD4+ T cells in the stage of BKPyV activation in KTRs.The re cipients were separated into 2 groups of BKPyV-active and nonactive KTRs (10 patients in each group) and were compared with 10 healthy control subjects. The viral load was evaluated by Taq-man quantitative real-time PCR. The frequency of different CD4+ T cell subsets was determined by analyzing markers such as CD45RO, CCR7, CD27, CD107a, perforin, and granzyme B using flow cytometry. The gene expression levels of transcription factors, including TBX21, GATA3, STAT3, and STAT6, contributing to CD4+ T cell activation, were also assessed. A significantly higher proportion in CCR7+CD27+CD45RO-CD4+ T cell (naive Tcell) subsets was detected in BKPyV-active KTRs compared to nonactive ones. A significant increase was detected in the frequency of CD107a+, perforin+, and granzyme B+ CD4+ T cells in the BKPyV-active group compared to the nonactive group. In CD4+ T cells of KTRs, the mRNA expression of TBX21 and GATA3 was significantly increased in KTRs without BKPyV reactivation compared to BKPyV-active ones. This investigation focused on the CD4+ T cell as an immunodominant T cell type with potential cytotoxicity. Based on these results, BKPyV may have a direct influence on the repertoire of CD4+ T cell subsets. Particularly, cytotoxic CD4+ T cells need further investigation to be considered as a therapeutic approach for BKPyV infection.

Development of a droplet digital PCR method for detection of porcine circovirus 4

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

Establishment of a triplex TaqMan quantitative real-time PCR assay for simultaneous detection of Cymbidium mosaic virus, Odontoglossum ringspot virus and Cymbidium ringspot virus.

Orchids are significant ornamental plants whose viral infection results in substantial economic damage. Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), and Cymbidium ringspot virus (CymRSV) represent three important and prevalent orchid viruses. The detection system proposed in this study uses a triplex TaqMan quantitative real-time PCR assay to identify CymMV, ORSV, and CymRSV in a simultaneous manner. We designed specific primers and probes for CymMV, ORSV, and CymRSV, with amplified sequences of 156 bp, 148 bp, and 145 bp, respectively. The minimum detection limit of the triplex qRT-PCR assay for CymMV and CymRSV was 1 copy/assay, and the minimum detection limit was 10 copies/assay for ORSV. The minimum stable detection limits for CymMV, ORSV, and CymRSV were 10, 102, and 102 copies/assay, respectively. Therefore, this system exhibited higher sensitivity (approximately 10 to 104-fold) than RT-PCR. The intra-and interassay CVs of Cq values are less than 0.55 and 0.95%, respectively, indicating that the triplex assay is highly reliable and accurate. In addition, 66 samples from five different orchid genera were analyzed using the established assay and gene chip. The detection results demonstrated that the triplex probe qRT-PCR demonstrated higher sensitivity than the gene chip, indicating that the triplex real-time PCR assay could be used for the detection of field samples. Our findings suggest that the triplex real-time RT-PCR detection system represents a rapid, simple, and accurate tool for detecting CymMV, ORSV, and CymRSV on orchids.

Development of a droplet digital PCR assay for detection of group A porcine rotavirus.

Group A porcine rotavirus (PoRVA) is an important pathogen of acute enteritis in piglets, which has caused severe economic losses in the pig industry worldwide. A convenient, sensitive and specific diagnosis method is an urgent requirement for the surveillance of the PoRVA circulating in clinical samples. In this study, a novel and convenient droplet digital PCR (ddPCR) for the detection of PoRVA was developed using the conserved region of the VP6 gene. The detection limit of ddPCR was 1.81 ± 0.14 copies/rection, ~10 times greater sensitivity than TaqMan real-time quantitative PCR (qPCR). Both ddPCR and qPCR assays exhibited good linearity and repeatability, and the established ddPCR method was highly specific for PoRVA. The results of clinical sample testing showed that the positivity rate of ddPCR (5.6%) was higher than that of qPCR (4.4%). Therefore, the newly developed ddPCR assay could be widely used in clinical diagnosis of PoRVA infections.

Parthenolide inhibits proliferation of cells infected with Kaposi's sarcoma-associated herpesvirus by suppression of the NF-κB signaling pathway.

The disease caused by Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the major causes of death of individuals with acquired immunodeficiency syndrome (AIDS). Development of anti-KSHV drugs is thus crucial. In this study, we investigated the effect of parthenolide (PTL) on the proliferation and NF-κB signaling pathway of KSHV-infected cells. iSLK.219 and KSHV-infected SH-SY5Y cells (SK-RG) were treated with PTL, TaqMan real-time quantitative PCR was used to determine the number of copies of the KSHV genome, and mRNA and protein expression of KSHV genes were analyzed by real-time PCR and immunocytochemistry. A cell viability test was used to measure cell proliferation, and flow cytometry was used to examine the effect of the drug on the cell cycle. Cyclin D1, CDK6, CDK4, and NF-κB-related proteins, including IKKβ, P-p65, and P-IKB-α, were detected by Western blot. The results showed that PTL altered the morphology of the cells, reduced the KSHV copy number, and suppressed the production of ORF50, K8.1, and v-GPCR mRNA and the LANA, ORF50, and K8.1 proteins. It blocked the G1 phase in iSLK.219 cells and decreased the levels of cyclin D1, CDK6, and CDK4 as well as the levels of NF-κB signaling proteins, including IKKβ, P-p65, and P-IKB-α. Together, these results suggest that PTL is a candidate drug that can decrease KSHV pathogenicity by suppressing cell proliferation and inhibiting the NF-κB signaling pathway in KSHV-infected cells.

Investigation of the Correlation Between the Polymorphism/Expression Level of RANTES and Its Receptor CCR5 Gene Promoter and Type 2 Diabetes Mellitus

This study aimed to explore relationship among RANTES -28 (rs2280788) C/G polymorphism or CCR5 59029 (rs1799987) A/G polymorphism, level of self-expression, and type 2 diabetes mellitus (T2DM). Clinical data were collected from 92 subjects with normal blood glucose (NC) and 97 patients with T2DM (DM). CCR5 levels on the surface of monocyte/lymphocyte and plasma RANTES levels were detected by flow cytometry. TaqMan real-time fluorescent quantitative PCR was used to detect genetic polymorphisms of RANTES rs2280788 and CCR5 rs1799987. There were no significant differences in frequencies of CCR5 rs1799987 genotype and A/G allele and frequencies of RANTES rs2280788 genotype and C/G allele, between subjects in NC and DM group (P > 0.05). Plasma RANTES level in DM group was significantly lower than NC group (P < 0.05), and difference came from patients with T2DM using insulin and subjects with normal blood glucose. CCR5 levels on the surface of monocytes and lymphocytes of patients in DM group were higher than NC group (P < 0.05). There was no significant difference in CCR5 level on the surface of monocytes and lymphocytes (or plasma RANTES level) among different genotypes of CCR5 rs1799987 (or RANTES rs2280788) (P > 0.05). RANTES level was positively correlated with age and TC and negatively correlated with diabetes course and HbA1c. CCR5 level on the surface of monocytes was positively correlated with drinking years, HbA1c, course of diabetes, and negatively correlated with TC. CCR5 on lymphocyte surface was positively correlated with diabetes course, smoking years, HbA1c, and negatively correlated with LDL, TC, HDL (P < 0.05). RANTES -28 (rs2280788) C/G polymorphism or CCR5 59029 (rs1799987) A/G polymorphism may not be associated with T2DM of Han nationality in Kunming and cannot affect RANTES and CCR5 expression. RANTES and CCR5 levels may be related to T2DM but may also be affected by age, blood lipids, HbA1c, diabetes course, drugs, and other factors.

Genetic diversity in Babesia bovis from southern Africa and estimation of B. bovis infection levels in cattle using an optimised quantitative PCR assay

Babesia bovis is a causal agent of bovine babesiosis, a disease which leads to mortality and morbidity and impacts the cattle industry worldwide. We amplified, cloned and sequenced the B. bovis merozoite surface antigen-2b (msa-2b) gene (∼940 bp) and the near full-length 18S rRNA gene (∼1600 bp) from cattle samples from South Africa and Mozambique to determine sequence variation between B. bovis parasites in the region. A TaqMan quantitative real-time PCR (qPCR) assay (18S rRNA gene) was optimised for the detection of B. bovis and estimation of parasitaemia in field samples from cattle from southern Africa. Phylogenetic analysis grouped the Msa-2b sequences in six clades and these were 59.7 to 99.6% identical to reference sequences. Sequence variation amongst B. bovis 18S rRNA sequences was found at 2 to 36 positions, and the sequences were 97 to 99% identical to published sequences. Mismatches between the B. bovis 18S rRNA sequences and a previously published qPCR forward primer (BoF) were observed; therefore, we developed a new forward primer (BoF2), and optimised the qPCR assay. Six 10-fold dilution series of B. bovis infected erythrocytes (2 × 108 to 2 × 103 infected red blood cells [iRBC]/ml) were analysed in triplicate in each of six separate qPCR runs, to determine the efficiency of the assay. The qPCR assay amplified the B. bovis 18S rRNA gene with 92.0 to 94.9% efficiency. The detection limit of the qPCR assay was approximately 6 iRBCs/μl. The performance of the optimised assay to diagnose B. bovis in field samples was assessed by testing DNA from 222 field samples of cattle from South Africa and Mozambique using three methods: the optimised qPCR assay, the reverse line blot (RLB) hybridisation assay, and the previously published qPCR assay. The detection rate of B. bovis using the optimised qPCR assay (31.1%, 69/222) was significantly higher (p<0.001) than both that using RLB (20.7%, 46/222) and the previously published qPCR assay (5.4%; 12/222). The B. bovis parasitaemia in samples from infected cattle ranged from 6 iRBCs/μl to 101,852 iRBCs/μl of blood. Our study revealed marked sequence variation between B. bovis parasites from southern Africa. The optimised qPCR assay will be useful in epidemiological studies and clinical diagnosis of B. bovis in southern Africa, and can be used to determine parasitaemia and potential carrier status in cattle populations, which is essential in the control of babesiosis.

Novel insights on the potential activity of propolis and wheat germ oil against chronic toxoplasmosis in experimentally infected mice

The use of apitherapy and natural herbal medicines for combating toxoplasmosis has garnered major attention from many researchers. However, there is no available information regarding the potential use of a combination of propolis and wheat germ oil (WGO) in the treatment of toxoplasmosis. In the present study, the potential effects of propolis, WGO, and their combination in the treatment of chronic toxoplasmosis in Swiss albino mice were investigated. Following induction of chronic toxoplasmosis, the potential antiparasitic effects of these substances were evaluated by parasitological assessment and by counting of Toxoplasma cysts. Additionally, the effects of the treatments on parasite loads were analyzed using TaqMan real-time quantitative PCR targeting the Toxoplasma P29 gene followed by investigation of the major histopathological changes in the brain, uterus, and kidney. Interestingly, the combination of propolis and WGO significantly (P ≤ 0.05) decreased the parasite burden in experimental animals compared with burdens seen in groups treated with propolis or WGO alone. Furthermore, the quantification of the DNA concentrations of Toxoplasma P29 gene after the treatment with propolis and WGO revealed a reduction in parasite load in treated groups versus the control group (infected untreated animals). Importantly, the severity of histopathological lesions was significantly (P ≤ 0.05) improved following treatment with propolis and WGO. Collectively, the present study indicated a potential novel role for propolis and WGO as an active apitherapy and natural herbal medication for treating chronic toxoplasmosis, combat the disease, and which could also help overcome the side effects of chemical drugs.

New design and optimization of an in-house quantitative TaqMan Real-Time PCR-based assay for the detection and monitoring of occult hepatitis B virus (genotype A-J) infection

PurposeHBV DNA quantification is used for individuals with uninterpretable serological tests, occult HBV infections, decreasing the window period of the disease, and treatment follow-up. Although there are commercial qPCR assays, they are expensive. In this study, we developed a highly sensitive quantitative TaqMan Real-Time PCR with an exogenous internal control to quantify HBV DNA in serum/plasma. MethodsA specific primer/probe set was designed for the S conserved region of various HBV genotypes. The primer/probe set was evaluated experimentally and in-silico. An exogenous internal control was included to monitor the effects of inhibitors. The standard plasmid was titrated using three different methods to prepare the seven standards for the assay. The functional characteristics of the in-house assay were evaluated using the standards. Two hundred clinical specimens were also tested. ResultsThe LOD of the in-house assay was 40 IU/mL, and the assay was linear from 3.26Log10 to 9.26Log10 IU/mL. The analytical and clinical sensitivity of the assay was 100% and 92.15%, respectively. The analytical and clinical specificity of the assay was 100% and 98.97%, respectively. The positive and negative predictive values of the assay were determined to be 98.94% and 92.38%, respectively. The highest coefficient of variation of the inter/intra-assay was 5.1%. The accuracy was close to 100% for all standards, and the correlation between the in-house assay and commercial kit AltoStar® PCR Kits 1.5 was remarkable. The results of the clinical samples using the standards titrated using AcroMetrix™ HBV Panel, Artus® HBV RG PCR Kit, and AltoStar® PCR Kits 1.5 were comparable (r ​= ​0.942, 0.951, 0.951). ConclusionsThe results indicate that the in-house assay is highly sensitive and specific, reproducible, and cost-benefit. Thus, it can be used to detect and quantify HBV DNA in research and clinical settings.

Development of a Novel RT-qPCR Detecting Method of Covert Mortality Nodavirus (CMNV) for the National Proficiency Test in Molecular Detection.

Covert mortality nodavirus (CMNV), the pathogen of viral covert mortality disease (VCMD), has caused serious economic losses of shrimp aquaculture in Southeast Asian countries and China in the past decade. In view of that the rapid and accurate laboratory detection of CMNV plays a major role in the effective control of the spread of VCMD. The national proficiency test (NPT) for the detection of covert mortality nodavirus (CMNV) started in China from 2021. In this study, a novel TaqMan real-time reverse transcription quantitative PCR (RT-qPCR) detection method for CMNV with higher sensitivity than previous reports was established based on specific primers and probe designing from the conserved regions of the CMNV coat protein gene for using molecular detection of CMNV in NPT. The optimized RT-qPCR reaction program was determined as reverse transcription at 54.9 °C for 15 min and denaturation at 95 °C for 1 min, followed by 40 cycles including denaturation at 95 °C for 10 s, and annealing and extension at 54.9 °C for 25 s. The detection limit of the newly developed RT-qPCR method was determined to be as low as 2.15 copies of CMNV plasmids template per reaction, with the correlation coefficient (R2) at above 0.99. The new method showed no cross reaction with the six common aquatic animal pathogens and could be finished in one hour, which represents a rapid detection method that can save 50% detection time versus the previously reported assay. The CMNV TaqMan probe based RT-qPCR method developed in present study supplies a novel sensitive and specific tool for both the rapid diagnosing and quantitating of CMNV in NPT activities and in the farmed crustaceans, and will help practitioners in the aquaculture industry to prevent and control VCMD effectively.

29P Plasma tumor-derived small extracellular vesicles microRNAs plus CA-125 objectively detect residual disease risk after surgical debulking in advanced ovarian cancer

29P Plasma tumor-derived small extracellular vesicles microRNAs plus CA-125 objectively detect residual disease risk after surgical debulking in advanced ovarian cancer