Fluorescent Quantitative PCR Assay Research Articles

Development and application of a quadruple RT-qPCR assay for the simultaneous detection of NoV GI, NoV GII, and HAV in bivalve shellfish.

To achieve rapid and simultaneous detection of NoV GI, NoV GII, and HAV, a quadruple real-time fluorescence quantitative PCR (RT-qPCR) assay was developed using MS2 bacteriophage as a process control virus. The quadruple RT-qPCR assay effectively detected NoV GI, NoV GII, HAV, and MS2 RNA with detection limits of 102 copies/μL, 103 copies/μL, 102 copies/μL, and 103 copies/μL, respectively, within 1 hour 50 minutes. The quadruple RT-qPCR assay could specifically detect NoV GI, NoV GII, HAV, and MS2 without cross-reactions with other common pathogens, demonstrating good reproducibility with intra-assay and inter-assay coefficients of variation all below 2.11%. In this study, 337 bivalve shellfish samples collected from various regions of Hebei Province were pretreated using the proteinase K-PEG 8000 precipitation-chloroform method, and viral nucleic acids were enriched and extracted from a volume of viral solution that was doubled. The developed quadruple RT-qPCR assay was used to detect NoV GI, NoV GII, and HAV in bivalve shellfish samples, and the positive rates were 19.88% (67/337), 20.47% (69/337), and 4.75% (16/337), respectively. In addition, mixed infections of NoV GI and NoV GII (10.68%, 36/337) and NoV GI and HAV (0.89%, 3/337) were observed. In all, 200 bivalve shellfish samples were randomly selected for the assay, and it was found that the total, positive, negative coincidence rates, and Kappa values of the quadruple RT-qPCR assay were 98.3%, 99.1%, 98.2%, and 0.945, respectively, compared with the single RT-qPCR assay. These results show that the developed quadruple RT-qPCR assay has comparable performance to the single RT-qPCR assay.IMPORTANCEFood-borne diseases caused by viral contamination have become a growing concern, and bivalve shellfish is a crucial source of infection, with many outbreaks of non-bacterial acute gastroenteritis associated with raw food or the use of undercooked shellfish such as oysters. As food contamination problems caused by NoV and HAV become more severe, it is important to study and establish a sensitive and efficient assay to simultaneously detect NoV and HAV by applying the MS2 process control virus for the protection of bivalve shellfish food safety and the monitoring of the above food-borne viral contamination. In addition, bivalve shellfish samples contain a large number of PCR inhibitors such as polysaccharides, lipids, and proteins, so optimization of the virus enrichment and extraction method is essential and is expected to provide a research basis for subsequent related experiments.

Casticin inhibits proliferation of non-small cell lung cancer cells by regulating glucose metabolism through suppression of HIF-1α

The study investigated the effect of casticin on the proliferation of non-small cell lung cancer(NSCLC) H322 cells and explored its molecular mechanism. Firstly, the cell counting kit-8(CCK-8) assay, colony formation assay, and EdU assay were used to detect the effect of casticin on the proliferation capacity of H322 cells under different concentrations and treatment durations. Then, glucose uptake, lactate production, extracellular pH, and oxygen consumption of H322 cells were measured before and after casticin treatment to analyze its impact on glycolysis in NSCLC H322 cells. Finally, real-time fluorescence quantitative PCR(RT-qPCR) and Western blot assays were performed to explore glycolysis-related molecules affected by casticin. The experiments showed that casticin inhibited the proliferation of NSCLC H322 cells in a dose-and time-dependent manner, with half-maximal inhibitory concentrations(IC_(50)) of 28.64 and 19.41 μmol·L~(-1) after 48 and 72 hours of treatment, respectively. Casticin also inhibited glucose uptake and lactate production in H322 cells, while increasing extracellular pH and oxygen consumption. Further investigation revealed that casticin inhibited the expression of glycolysis-related molecules, including glucose transporter 1(GLUT1), hexokinase 2(HK2), aldolase A(ALDOA), pyruvate kinase M2(PKM2), and hypoxia-inducible factor-1α(HIF-1α). Overexpression of HIF-1α was found to reverse the inhibitory effects of casticin on H322 cell proliferation and glycolysis. These findings suggest that casticin may regulate cellular glycolysis by inhibiting the expression of HIF-1α, thereby inhibiting the proliferation of NSCLC H322 cells. This study identifies a potential drug for the treatment of NSCLC and provides a direction for further research.

Development and application of a quadruplex TaqMan real-time fluorescence quantitative PCR assay for four porcine digestive pathogens.

Salmonella, Escherichia coli, Lawsonella intracellularis, and Brachyspira hyodysenteriae are the primary pathogens responsible for gastrointestinal diseases in pigs, posing a significant threat to the health and productivity of pig production systems. Pathogen detection is a crucial tool for monitoring and managing these infections. We designed primers and probes targeting the invA gene of Salmonella, the 23S rRNA gene of Escherichia coli, the aspA gene of Lawsonella intracellularis, and the nox gene of Brachyspira hyodysenteriae. We developed a quadruplex TaqMan real-time quantitative PCR assay capable of simultaneously detecting these four pathogens. This assay demonstrated high sensitivity, with detection limits of 100 copies/μL for the recombinant plasmid standards pEASY-23S rRNA, pEASY-aspA, and pEASY-nox, and 10 copies/μL for pEASY-invA. The standard curves exhibited excellent linearity (R2 values of 0.999, 0.999, 1, and 0.998, respectively) and high amplification efficiencies (93.57%, 94.84%, 85.15%, and 81.81%, respectively). The assay showed high specificity, with no cross-reactivity detected against nucleic acids from Streptococcus suis, porcine epidemic diarrhoea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), Pasteurella multocida, Clostridium perfringens, Gracilaria parapsilosis, porcine delta coronavirus (PDCoV), porcine group A rotavirus (GARV), and porcine teschovirus (PTV). The assay also exhibited excellent repeatability, with inter- and intra-assay coefficient of variation (CV) ranging from 0.15% to 1.12%. High concentrations of nucleic acids did not interfere with the detection of low concentrations, ensuring robust performance in complex samples. Among 263 diarrhoeic samples, the assay detected Salmonella in 23.95%, Escherichia coli in 26.24%, Lawsonella intracellularis in 33.84%, and Brachyspira hyodysenteriae in 22.43%. This quadruplex TaqMan qPCR assay offers a rapid, sensitive, and specific tool for the simultaneous detection of Salmonella, Escherichia coli, Lawsonella intracellularis, and Brachyspira hyodysenteriae in pigs.

One-Step Multiplex Real-Time Fluorescent Quantitative Reverse Transcription PCR for Simultaneous Detection of Four Waterfowl Viruses.

Duck Tembusu virus (DTMUV), duck hepatitis virus (DHV), Muscovy duck reovirus (MDRV), and Muscovy duck parvovirus (MDPV) represent four emergent infectious diseases impacting waterfowl, which can be challenging to differentiate due to overlapping clinical signs. In response to this, we have developed a one-step multiplex real-time fluorescence quantitative reverse transcription PCR (qRT-PCR) assay, capable of simultaneously detecting DTMUV, DHV, MDRV, and MDPV. This method exhibits high specificity, avoiding cross-reactivity with other viruses such as Fowl adenoviruses (FADV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Haemophilus paragallinarum (Hpg), duck circovirus (DUCV), goose astrovirus (GoAstV), and mycoplasma gallisepticum (MG). The limit of detection (LOD) established for DTMUV, DHV, MDRV, and MDPV was determined to be 27 copies/μL. In the repeatability test, the intra-assay and inter-assay coefficients of variation (CVs) of the recombinant plasmid standard were less than 2%. Utilizing this method, we analyzed 326 clinical specimens sourced from Guangxi over the period spanning October 2021 through December 2023, yielding promising and precise outcomes. The qRT-PCR method established herein exhibits commendable specificity, sensitivity, and repeatability. Furthermore, it boasts a high clinical detection rate, making it a highly effective tool for diagnosing these pathogenic agents in waterfowl.

Development and application of a quadruplex TaqMan fluorescence quantitative PCR typing method for Streptococcus suis generalis, type 2, type 7 and type 9.

Streptococcus suis (SS) is one of the most important pathogens causing major economic losses in the global pig farming industry and is a serious threat to public health safety. It has multiple serotypes, with poor cross-protection between serotypes, and effective typing methods are lacking. In this study, a quadruplex TaqMan fluorescence quantitative PCR assay that can differentiate between Streptococcus suis types 2, 7 and 9 was developed using the gdh gene, a generic gene for Streptococcus suis, and cps2J, cps7H and cps9J, genes encoding podocarp-associated genes for types 2, 7 and 9, respectively, as targets. The method is specific enough to accurately type Streptococcus suis pigmentosus without detecting non-target pathogens (Escherichia coli, Pasteurella multocida, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae and etal). The sensitivity was high, with a minimum lower detection line of 10 copies for P-SS and P-SS9, and 100 copies for P-SS2 and P-SS7. The standard curves generated showed good linearity with R2 of 0.999, 0.999, 0.997 and 0.998 respectively. The repeatability was good, with coefficients of variation between batch to batch and batch to batch tests ranging from 0.21% to 1.10%. Testing of 156 samples yielded 68 positive and 88 negative samples, of which the positive rate of SS was 5.77% (9/156), SS2 was 20.51% (32/156), SS7 was 8.33% (13/156) and SS9 was 9.6% (15/156), which was in line with the existing fluorescent quantitative PCR assay of 93.75%~100%, which was higher than the detection rate of conventional PCR. The quadruplex TaqMan fluorescence quantitative PCR method of Streptococcus suis generic, type 2, 7 and 9 established in this study can accurately differentiate the three serotypes of Streptococcus suis that currently have high prevalence and pathogenicity, which is of great importance for accurate clinical prevention and treatment, epidemiological investigation and vaccine development.

Establishment and application of a duplex fluorescent quantitative PCR assay for H9N2 subtype avian influenza virus and infectious bronchitis virus.

The H9N2 subtype of avian influenza virus (AIV) and infectious bronchitis virus (IBV) are important avian viruses that cause respiratory symptoms in poultry, and can form mixed infections. In this study, primers and probes were designed based on the HA gene of H9N2 and the 5' noncoding region of IBV, respectively, and a fluorescent quantitative RT-PCR assay was established for simultaneous detection of these two pathogens. The reaction system and conditions were optimized. The method only detected AIV subtype H9N2 and IBV and no other viruses, confirming its high specificity. The assay detected 13.5 copies/μL and 1.66 copies/μL of H9N2 and IBV in clinical samples, respectively. The coefficients of variation for intra- and interassay repeatability were < 3%. The established method was used to analyze 254 clinical samples (oropharyngeal and cloacal swabs) from Hubei Province, China; 98.82% were positive for both pathogens. In summary, a duplex fluorescent quantitative RT-PCR method capable of simultaneously detecting AIV subtype H9N2 and IBV was established. It is specific, sensitive, and reproducible, and can be used for diagnosis of a variety of clinical samples. It provides a technological means for the rapid and simultaneous detection of both pathogens, and thus can facilitate clinical diagnosis and epidemiological investigations.

Development and Application of a TaqMan RT-qPCR for the Detection of Foot-and-Mouth Disease Virus in Pigs.

The global livestock industry is facing a serious threat from a widespread foot-and-mouth disease virus (FMDV) epidemic. The timely detection of FMDV can significantly mitigate its harmful effects. This study aimed to establish and evaluate a TaqMan fluorescence quantitative PCR assay to assess its sensitivity, specificity, reproducibility, and stability. The standard curve equation range is 6.43 × 109-6.43 × 101 copies/µL, with an R2 value of 0.996 and a standard curve equation of y = -3.586x + 36.245. The method successfully detected 64.3 copies/µL of the target gene for FMDV and exhibited high specificity for FMDV. Repeatability tests demonstrated low coefficients of variation within and between groups (<2%), indicating good reproducibility. The clinical samples analyzed using this method showed results consistent with those of the SYBR Green I RT-qPCR assay, confirming the reliability of the method. Overall, the developed test method displayed high sensitivity, specificity, reproducibility, and stability, making it suitable for the rapid diagnosis of foot-and-mouth disease in clinical settings.

Ruhao Dashi granules exert therapeutic effects on H1N1 influenza virus infection by altering intestinal microflora composition.

Antiviral medications for influenza could be ineffective due to the emergence of resistant influenza virus strains. Ruhao Dashi (RHDS) granules possess anti-inflammatory and antibacterial effects. The present study aimed to determine the efficacy of RHDS granules in treating influenza-infected mice and the mechanism underlying this treatment as well as its effect on the intestinal flora composition of the infected mice. The HPLC-UV method was used to identify the active components of RHDS granules. ICR mice were infected with influenza A virus (IAV) H1N1 subtype through a nasal drip. After the influenza mice model was successfully established, the pathological changes in the lungs were observed for 5 days after gavage treatment with 0.9% sterile saline and low, medium, and high doses (0.07, 0.14, and 0.28 g/mL, respectively) of RHDS granules. The serum levels of the cytokines IL-6 and TNF-α and sIgA were detected by ELISA. Real-time fluorescence quantitative PCR and western blotting assay were performed to determine the expression levels of the tight junction (TJ) proteins claudin-1, occludin, and zonula occludens-1 (ZO-1) in colon tissues. Furthermore, 16S rRNA gene sequencing of feces samples was conducted to assess the effect of RHDS granules on the gut microbiota. RHDS granules exerted a protective effect on the lung tissues of IAV-infected mice; moreover, the granules reduced the synthesis of proinflammatory cytokines and increased the relative expression levels of claudin-1, occludin, and ZO-1 in colon tissues. Furthermore, RHDS granule treatment increased the relative abundance of Lactobacillus, Akkermansia, and Faecalibaculum and decreased the relative abundance of Muribaculaceae; thus, RHDS granules could stabilize the intestinal microbiota to some extent. RHDS granules exert a therapeutic effect on IAV-infected mice probably by modifying the structural composition of their intestinal microbiota.

Bone Marrow Mesenchymal Stem Cells-Derived Extracellular Vesicle miR-208a-3p Alleviating Spinal Cord Injury via Regulating the Biological Function of Spinal Cord Neurons.

We aim to explore the potential mechanism of bone marrow mesenchymal stem cells-derived extracellular vesicles (BMSCs-Exo) in improving spinal cord injury (SCI). Thirty male 12-week specific pathogen-free (SPF) Sprague-Dawley (SD) rats were used to construct SCI model invivo. Ten male 12-week SPF SD rats were used to extract BMSCs. The Basso, Beattie, Bresnahan (BBB) score was used to evaluate the motor function of rats. Real-time fluorescence quantitative PCR (RT-PCR), western blot (WB), and double luciferase assay were used to explore the regulation between rno-miR-208a-3p and Cdkn1a (p21) in BMSCs. Primary spinal cord neurons were treated with lipopolysaccharide (100 ng/mL) for 30 min to mimic SCI in vitro. Compared with the model group (14 scores), BMSCs-Exo increased BBB score (19 scores) in SCI rats. Compared with the sham group, Cdkn1a was upregulated, whereas rno-miR-208a-3p was downregulated in the model group. However, compared with the model group, Cdkn1a was downregulated, whereas rno-miR-208a-3p was upregulated in the BMSCs-Exo group. In addition, rno-miR-208a-3p inhibited the expression of Cdkn1a via direct binding way. BMSCs-Exo-rno-miR-208a-3p promoted the proliferation of primary spinal neurons via inhibiting apoptosis in vitro. Moreover, BMSCs-Exo-rno-miR-208a-3p promoted cyclin D1, CDK6, and Bcl-2 and inhibited Bax expression in a cell model of SCI. In conclusion, BMSCs-Exo-carried rno-miR-208a-3p significantly protects rats from SCI via regulating the Cdkn1a pathway.

Distribution and seasonal fluctuation of visceral leishmaniasis vectors sandflie in Lüliang City of Shanxi Province in 2023

To investigate the distribution and seasonal fluctuations of visceral leishmaniasis vectors sandflies in Lüliang City, Shanxi Province, so as to provide insights into assessment of the visceral leishmaniasis transmission risk and formulation of visceral leishmaniasis control measures. A total of 12 natural villages were sampled from Shilou County, Lishi District, Lanxian County, Linxian County and Wenshui County in Lüliang City, Shanxi Province from June to September, 2023, and sandflies were captured using light traps from 7 breeding habitats, including farmers' houses, sheep pens, cattle pens, chicken coops, pig pens, mule and horse pens, and loess-cave dwellings. Following morphological identification of the sandfly species, the distribution of sandflies and the seasonal fluctuations of the sandfly density were analyzed. In addition, the Leishmania was detected in sandflies using a real-time fluorescence quantitative PCR assay. A total of 2 831 sandflies were captured with 156 light traps in Lüliang City from June to September, 2023, including 2 638 female sandflies (93.18%) and 193 male sandflies (6.82%), and the average density was 16.91 sandflies/(light-night). The seasonal fluctuations of the sandfly density all appeared a unimodal distribution in all survey sites, and the sandfly density peaked in July and then declined rapidly. Among all types of breeding habitats, the greatest sandfly density was found in sheep pens [39.04 sandflies/(light-night)]. In addition, 4.08% (2/49) of the sandfly samples were tested positive for Leishmania nucleic acid as revealed by the real-time fluorescence quantitative PCR assay. Sandflies were widely distributed in Lüliang City, Shanxi Province in 2023, and the peak of the sandfly density was observed in July, which had a visceral leishmaniasis transmission risk. Intensified surveillance of visceral leishmaniasis and sandfly vectors is required and targeted vector control is recommended.

Duplex Real-Time Fluorescent Quantitative PCR Assays for the Detection of Toxigenic Microcystis Genotypes Based on SNP/InDel Variation in mcy Gene Cluster.

In this study, the toxigenic characteristics of 14 strains of Microcystis were analyzed, and single nucleotide polymorphism (SNP) and insertion/deletion (InDel) loci in microcystinsynthetase (mcy)geneclusters were screened. Based on SNP and InDel loci associated with the toxigenic characteristics, primers andTaqMan or Cycling fluorescent probes were designed to develop duplex real-time fluorescent quantitative PCR (FQ-PCR) assays. After evaluating specificity and sensitivity, these assays were applied to detect the toxigenic Microcystis genotypes in a shrimp pond where Microcystis blooms occurred. The results showed a total of 2155 SNP loci and 66 InDel loci were obtained, of which 12 SNP loci and 5 InDel loci were associated with the toxigenic characteristics. Three duplex real-time FQ-PCR assays were developed, each of which could quantify two genotypes of toxigenic Microcystis. These FQ-PCR assays were highly specific, and two Cycling assays were more sensitive than TaqMan assay. In the shrimp pond, six genotypes of toxigenic Microcystis were detected using the developed FQ-PCR assays, indicating that above genotyping assays have the potential for quantitative analysis of the toxigenic Microcystis genotypes in natural water.

Establishment of an A/T-Rich Specifically MGB Probe digital droplet PCR Assays Based on SNP for Brucella wild strains and vaccine strains

In recent years, immunization with the S2 live-attenuated vaccine has been recognized as the most economical and effective strategy for preventing brucellosis in Inner Mongolia, China. However, there are still challenges related to vaccine toxicity and the inability to distinguish between vaccine immunization and natural infection. Therefore, in this study, we developed a digital droplet polymerase chain reaction (ddPCR) assay based on single-nucleotide polymorphism (SNP) loci to identify wild Brucella strains and S2 vaccine strains. The assay demonstrated excellent linearity (R2> 0.99) with a lower detection limit of 10 copies/µL for both wild and vaccine strains. Additionally, the ddPCR assay outperformed the real-time fluorescent quantitative PCR (qPCR) assay in screening 50 clinical samples. We have established an effective and highly sensitive ddPCR assay for Brucella, providing an efficient method for detecting and differentiating wild strains of Brucella from the S2 vaccine strain.

Porphyromonas gingivalis persisters induce the immuno-inflammatory responses in macrophages by upregulating the forkhead box1 signaling pathway

Objective: To investigate the effects of Porphyromonas gingivalis (Pg) persisters (Ps) on immuno-inflammatory responses in macrophages, and to explore the underlying mechanisms. Methods: Pg cells were cultured to the stationary phase (72 h), and subsequently treated by high concentration of metronidazole at 100 mg/L, amoxicillin at 100 mg/L and the combination of them for different time period, named as metronidazole group, amoxicillin group and (metronidazole+amoxicillin) group. Pg cells without treatment were used as Blank control. The survival profile of PgPs cells was measured by colony-forming unit assay. The living state of PgPs was observed by Live/Dead staining. Then, Pg and metronidazole-treated PgPs (M-PgPs) were used to treat macrophages, named as Pg group and M-PgPs group. Transmission electron microscopy (TEM) was used to observe the bacteria in the macrophages. The expression levels of proinflammatory cytokines in macrophages were determined by real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay. The location of forkhead box transcription factor 1 (FOXO1) was detected by confocal immunofluorescence microscopy. After inhibiting or enhancing the FOXO1 expressions using inhibitors (Fi) or activators (Fa) respectively, the macrophages were treated with Pg and M-PgPs, divided as Blank group, Pg group, M-PgPs group, Fi group, (Fi+Pg) group, (Fi+M-PgPs) group, Fa group, (Fa+Pg) group and (Fa+M-PgPs) group. Then, the expression pattens of proinflammatory cytokines were assessed. Results: Remarkable number of lived PgPs was observed, both in planktonic culture and Pg biofilms either treated with metronidazole, amoxicillin or both, and those persisters could form new colonies. Pg and M-PgPs were able to enter into the macrophages and the protein expression levels of interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α (TNF-α) [Pg group: (2 392±188), (162±29), (5 558±661), (789±155) μg/L; M-PgPs group: (2 415±420), (155±3), (5 732±782), (821±176) μg/L] were significantly upregulated than those in Blank group [(485±140), (21±9), (2 332±87), (77±7) μg/L] (P<0.01). Moreover, Pg and M-PgPs could facilitate the nuclear translocation and accumulation of FOXO1. In addition, the relative mRNA expression levels of FOXO1, B-cell lymphoma 6 and Krüppel-like factor 2 were upregulated when compared to Blank group (P<0.05). Furthermore, the protein expression levels of IL-1β, IL-6, IL-8 and TNF-α in Fi+Pg group [(1 081±168), (70±8), (1 976±544), (420±47) μg/L] were remarkably lower than Pg group [(4 411±137), (179±6), (5 161±929), (934±24) μg/L] (P<0.05). Similarly, the protein expression levels of IL-1β, IL-6, IL-8 and TNF-α in Fi+M-PgPs group [(1 032±237), (74±10), (1 861±614), (405±32) μg/L] were remarkably lower than M-PgPs group [(4 342±314), (164±17), (4 438±1 374), (957±25) μg/L] (P<0.05). On the contrary, the protein expression levels of IL-1β, IL-6, IL-8 and TNF-α in Fa+Pg group [(8 198±1 825), (431±28), (8 919±650), (2 186±301) μg/L] and Fa+M-PgPs group [(8 159±2 627), (475±26), (8 995±653), (2 255±387) μg/L] were significantly higher than Pg group and M-PgPs group, respectively (P<0.05). Conclusions: PgPs are highly tolerant to metronidazole and amoxicillin. The M-PgPs could enhance the immuno-inflammatory responses in macrophages by upregulating the FOXO1 signaling pathway, while this effect exhibits no significant difference with Pg.

A duplex fluorescent quantitative PCR assay to distinguish the genotype I, II and I/II recombinant strains of African swine fever virus in China.

African swine fever (ASF) is a severe, hemorrhagic, and highly contagious disease caused by the African swine fever virus (ASFV) in both domestic pigs and wild boars. In China, ASFV has been present for over six years, with three genotypes of strains prevalent in field conditions: genotype I, genotype II, and genotype I/II recombinant strains. In order to differentiate among these three ASFV genotypes, a duplex fluorescent quantitative PCR method was established using specific probes and primers designed based on viral genes MGF_110-1L and O61R from ASFV strains reported in the GenBank database. Following optimization of reaction conditions, a duplex fluorescent quantitative PCR method was successfully developed. This method demonstrated no cross-reactivity with porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine circovirus 2 (PCV2), porcine circovirus 3 (PCV3), highlighting its specificity. Sensitivity analysis revealed that the limits of detection (LODs) of this method were 2.95 × 10-1 copies/μL for the MGF_110-1L gene and 2.95 × 100 copies/μL for the O61R gene. The inter- and intra-group coefficients of variation were both <1%, indicating high reproducibility. In summary, the establishment of this duplex fluorescent quantitative PCR method not only addresses the identification of the ASFV recombinant strains but also allows for simultaneous identification of the three epidemic genotype strains.

Dynamic observation on capillarization of liver sinusoidal endothelial cells induced by Echinococcus multilocularis infection

To investigate the capillarization of liver sinusoidal endothelial cells (LSECs) and its association with hepatic fibrosis during the development of alveolar echinococcosis, so as to provide the basis for unraveling the mechanisms underlying the role of LSEC in the development and prognosis of hepatic injuries and hepatic fibrosis caused by alveolar echinococcosis. Forty C57BL/6 mice at ages of 6 to 8 weeks were randomly divided into a control group and 1-, 2- and 4-week infection groups, of 10 mice in each group. Each mouse in the infection groups was intraperitoneally injected with 2 000 Echinococcus multilocularis protoscoleces, while each mouse in the control group was given an equal volume of phosphate-buffered saline using the same method. All mice were sacrificed 1, 2 and 4 weeks post-infection and mouse livers were collected. The pathological changes of livers were observed using hematoxylin-eosin (HE) staining, and hepatic fibrosis was evaluated through semi-quantitative analysis of Masson's trichrome staining-positive areas. The activation of hepatic stellate cells (HSCs) and extracellular matrix (ECM) deposition were examined using immunohistochemical staining of α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1A1), and the fenestrations on the surface of LSECs were observed using scanning electron microscopy. Primary LSECs were isolated from mouse livers, and the mRNA expression of LSEC marker genes Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf was quantified using real-time fluorescence quantitative PCR (qPCR) assay. Destruction of local liver lobular structure was observed in mice 2 weeks post-infection with E. multilocularis protoscoleces, and hydatid cysts, which were surrounded by granulomatous tissues, were found in mouse livers 4 weeks post-infection. Semi-quantitative analysis of Masson's trichrome staining showed a significant difference in the proportion of collagen fiber contents in mouse livers among the four groups (F = 26.060, P < 0.001), and a higher proportion of collagen fiber contents was detected in mouse livers in the 4-week infection group [(11.29 ± 2.58)%] than in the control group (P < 0.001). Immunohistochemical staining revealed activation of a few HSCs and ECM deposition in mouse livers 1 and 2 weeks post-infection, and abundant brown-yellow stained α-SMA and COL1A1 were deposited in the lesion areas in mouse livers 4 weeks post-infection, which spread to surrounding tissues. Semi-quantitative analysis revealed significant differences in α-SMA (F = 7.667, P < 0.05) and COL1A1 expression (F = 6.530, P < 0.05) in mouse levers among the four groups, with higher α-SMA [(7.13 ± 3.68)%] and COL1A1 expression [(13.18 ± 7.20)%] quantified in mouse livers in the 4-week infection group than in the control group (both P values < 0.05). Scanning electron microscopy revealed significant differences in the fenestration frequency (F = 37.730, P < 0.001) and porosity (F = 16.010, P < 0.001) on the surface of mouse LSECs among the four groups, and reduced fenestration frequency and porosity were observed in the 1-[(1.22 ± 0.48)/μm2 and [(3.05 ± 0.91)%] and 2-week infection groups [(3.47 ± 0.10)/μm2 and (7.57 ± 0.23)%] groups than in the control group (all P values < 0.001). There was a significant difference in the average fenestration diameter on the surface of mouse LSECs among the four groups (F = 15.330, P < 0.001), and larger average fenestration diameters were measured in the 1-[(180.80 ± 16.42) nm] and 2-week infection groups [(161.70 ± 3.85) nm] than in the control group (both P values < 0.05). In addition, there were significant differences among the four groups in terms of Stabilin-1 (F = 153.100, P < 0.001), Stabilin-2 (F = 57.010, P < 0.001), Ehd3 (F = 31.700, P < 0.001), CD209b (F = 177.400, P < 0.001), GATA4 (F = 17.740, P < 0.001), and Maf mRNA expression (F = 72.710, P < 0.001), and reduced mRNA expression of Stabilin-1, Stabilin-2, Ehd3, CD209b, GATA4 and Maf genes was quantified in three infection groups than in the control group (all P values < 0.001). E. multilocularis infections may induce capillarization of LSECs in mice, and result in a reduction in the expression of functional and phenotypic marker genes of LSECs, and capillarization of LSECs occurs earlier than activation of HSC and development of hepatic fibrosis.

Preliminary study on the effect of Echinococcus multilocaris on phenotypic transformations of glucose metabolism and polarization types in macrophages

To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with E. multilocularis protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays. There were significant differences among the four groups in terms of Arginase-1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages-derived C-C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin-like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL-6 (F = 1 823.00, P < 0.000 1), IL-10 (F = 291.70, P < 0.000 1), IL-1β (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)-α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)-β mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL-10, IL-1β, TNF-α and TGF-β in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL-6 mRNA expression in the BMDM-CF co-culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL-6 and TNF-α and higher TGF-β mRNA expression (both P values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both P values < 0.05). Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.

Blueberry anthocyanins improve liver fibrosis by regulating NCOA4 ubiquitination through TRIM7 to affect ferroptosis of hepatic stellate cells.

This study aims to investigate the role and molecular mechanism of anthocyanin in improving liver fibrosis through ferroptosis, providing a basis for drug development and targeted therapy. In this study, a mouse model of liver fibrosis was established using CCl4, and the anthocyanin treatment groups were administered 100 mg/kg anthocyanin daily via gavage. Furthermore, real-time fluorescent quantitative PCR (qRT-PCR), Western blotting (WB), and enzyme-linked immunosorbent assay were used to assess liver fibrosis indicators and liver injury markers. Histopathological methods were used to confirm the morphology of liver injury in different treatment groups. The effects of anthocyanins on ferroptosis markers, NCOA4 and FTH1 expression, were examined through qRT-PCR, WB, and Co-IP. Confocal microscopy was used to validate the colocalization of ferritin and lysosomes. A differential expression model of TRIM7 was constructed to verify its impact on the progression of liver fibrosis. The present study demonstrates the hepatoprotective effects of anthocyanins in liver fibrosis, highlighting their ability to enhance hepatic stellate cell (HSC) ferroptosis and regulate ferritin autophagy. Moreover, TRIM7 is identified as a key mediator of anthocyanin-induced regulation of hepatic stellate cells activation for liver fibrosis treatment through modulation of ferroautophagy. Mechanistic investigations further reveal that TRIM7 exerts its influence on the process of ferroautophagy by controlling NCOA4 ubiquitination. Our study discovered that anthocyanins could improve liver fibrosis by regulating NCOA4 ubiquitination through TRIM7, thereby affecting hepatic stellate cells' ferroptosis levels.NEW & NOTEWORTHY This was the first study to demonstrate that anthocyanins can improve the progression of liver fibrosis by promoting hepatic stellate cell (HSC) ferroptosis. Anthocyanins could affect the content of Fe2+ by promoting ferroautophagy in HSCs, thereby promoting the level of ferroptosis. This study demonstrates for the first time that anthocyanins can inhibit the expression of TRIM7 and then affect the ubiquitination of NCOA4 to regulate the level of ferritin autophagy and ferroptosis.

Comparison of dye-based and probe-based RT-LAMP in detection of canine astrovirus.

A rapid and sensitive assay is essential for reliable surveillance and diagnosis of canine astrovirus (CaAstV). In this study, two real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays with high sensitivity, rapidity, and reliability were developed using fluorescence dye and FRET-based assimilating probes for real-time detection of CaAstV. These assays specifically amplified the ORF2 gene of CaAstV and did not amplify any sequences from canine enterovirus. The limit of detection (LOD) of both the probe-based and dye-based RT-LAMPs was 100 copies/μL. Fluorescence signals were generated within 30 min for the lowest concentration of a standard RNA sample, which was significantly faster than that achieved by real-time fluorescence quantitative PCR (qRT-PCR) assay. When clinical samples were tested, the positive and negative agreement of the dye-based RT-LAMP assay with qRT-PCR was 87.5% (14/16) and 93.55% (29/31), respectively. The positive and negative agreement of the probe-based RT-LAMP assay with qRT-PCR was 94.11% (16/17) and 96.55% (28/29), respectively. The RT-LAMP assays developed in this study showed strong potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of CaAstV in clinical samples.

Effects ofCallicarpa nudifloraGranules on the Proliferation and Apoptosis of Uterine Fibroid Cells

This research was aimed to discuss and understand the effects and mechanisms of action ofCallicarpa nudifloragranules on proliferation and apoptosis of uterine leiomyoma (UL) cells. Firstly, normal uterine myometrium (UM) and UL tissues were collected, and the levels of p-Akt and Phosphatase and Tensin Homolog (PTEN) in UL tissues were detected using immunohistochemistry. Next, the UL cells were successfully obtained using enzymatic digestion, and their identification was performed using alpha-smooth muscle actin (α-actin) immunocytochemistry. Specifically, the cells were grouped into four: a control group (CG), a low-dose group (LDG, 50 mg/LCallicarpa nudiflorasolution), a medium-dose group (MDG, 100 mg/LCallicarpa nudiflorasolution), and a high-dose group (HDG, 200 mg/LCallicarpa nudiflorasolution). Moreover, the proliferation of UL cells was assessed using the thiazolyl blue (MTT) assay, while cell apoptosis was analyzed using flow cytometry (FCT). Real-time fluorescent quantitative PCR (fq-PCR) and Western blot assay (WBA) were utilized to determine the PAI-1, P38, TGF-β1, E-cadherin, and Vimentin in UL cells. The results revealed that the positive rate (PR) of p-Akt in the UL tissues was much higher to that in normal UM tissues (P&lt; 0.001). More than 90% of UL cells were positive forα-actin. The viabilities of UL cells in theCallicarpa nudifloratreatment groups were greatly weakened to that of untreated cells (P&lt; 0.05). Viability of UL cells in the HDG group was the lowest, showing a great difference withP&lt; 0.01 to the LDG group and that withP&lt; 0.05 to the MDG group, while that between the MDG and LDG groups exhibited a great difference withP&lt; 0.05. AR of UL cells in CG group was sharply lower to that in theCallicarpa nudifloratreatment groups, showing great differences withP&lt; 0.05,P&lt; 0.01, andP&lt; 0.001, respectively. AR of UL cells in HDG group was higher to the LDG group (P&lt; 0.01) and MDG group (P&lt; 0.05), and that in LDG group was lower and exhibited a great difference withP&lt; 0.05 to the MDG group. The HDG, LDG, and MDG groups exhibited greatly lower TGF-β1, PAI-1, and P38 to the CG group (P&lt; 0.05). In the HDG group, the TGF-β1, PAI-1, P38, and Vimentin levels were greatly lower and presented a great difference withP&lt; 0.01 to those in the CG group and LDG group. Additionally, E-cadherin in UL cells was elevated in the LDG and MDG groups to CG group, showingP&lt; 0.05 andP&lt; 0.01, respectively. Such findings indicated that theCallicarpa nudifloragranules can suppress proliferation of UL cells and promote their apoptosis, which may be associated with the TGF-β1/P38/PAI-1 singling pathway (SPW).

Diagnostic Efficacy of Plasma-Based Real-Time PCR for Schistosomiasis Japonica in Mice before and after Treatment with Praziquantel.

The prevalence of schistosomiasis japonica in China is now characterized by a low epidemic rate and low-intensity infections. Some diagnostic methods with high sensitivity and specificity are urgently needed to better monitor this disease in the current situation. In this study, the detection efficacy of a real-time fluorescent quantitative PCR (qPCR) assay was assessed for schistosomiasis japonica in mice, and before and after treatment with praziquantel (PZQ). Our results showed that the sensitivity of the qPCR was 99.3% (152/153, 95% CI: 96.41-99.98%) and its specificity was 100% (77/77, 95% CI: 95.32-100%) in mice infected with different numbers of Schistosoma japonicum. After the oral administration of PZQ, mice infected with 10 cercariae or 40 cercariae were all Schistosoma japonicum-negative 6 weeks after treatment. However, the negativity rates on a soluble egg antigen (SEA)-based enzyme-linked immunosorbent assay (ELISA) were only 34.8% (8/23, 10 cercariae group) and 6.7% (1/15, 40 cercariae group) at the sixth week after PZQ treatment. These results demonstrated that the qPCR method had good sensitivity and specificity, and suggested that its sensitivity correlated with the infection intensity in mice. Moreover, this method had better potential utility for evaluating the treatment efficacy of PZQ in schistosome-infected mice than SEA-based ELISA.