Fluorescence Quantitative Method Research Articles

Establishment and application of quadruple fluorescence quantitative RT-PCR method for the identification of waterfowl astrovirus

Avian astrovirus can infect a variety of poultry species and cause viral diarrhea, with a wide epidemic range strong pathogenicity and a high incidence. Among them, Duck astrovirus 3(DAstV-3), Duck astrovirus 4(DAstV-4), Goose astrovirus 1(GoAstV-1) and Goose astrovirus 2(GoAstV-2) are four types of astroviruses newly discovered in waterfowl in recent years. In order to realize the rapid detection of these four kinds of waterfowl stellate viruses, specific primers and probes were engineered to target a highly conserved region of ORF1b gene of DAstV-3, GoAstV-1 and GoAstV-2 and the ORF2 gene of DAstV-4, and a quadruple fluorescence quantitative RT-PCR method was developed. The results indicate that the method established in this study has good specificity and no cross reactivity with other pathogens. This method can detect viruses with a minimum concentration of 1 × 101 copies/μL for DAstV-4, GoAstV-1 and GoAstV-2, respectively, while the minimum concentration for DAstV-3 is 1 × 102 copies/μL. Compared with the routinely used RT-PCR method, the limit of detection by the multiplex RT-PCR lower. Both intra- and inter-assay variability tests revealed excellent reproducibility. This method was then used to analyze 269 field samples, and the results were verified by genome sequencing. In conclusion, this study presents a sensitive, accurate, and specific method for detecting DAstV-3, DAstV-4, GoAstV-1, and GoAstV-2 in a single reaction, enabling the monitoring and differential diagnosis of these four types of waterfowl astroviruses.

Remazolam affects the phenotype and function of astrocytes to improve traumatic brain injury by regulating the Cx43

PurposeTo explore the mechanism by which Remazolam affects the phenotype and function of astrocytes to improve traumatic brain injury (TBI). MethodsThe oxygen -glucose deprivation/recovery (OGD/R) cell model was constructed to simulate the pathological state of astrocytes in a TBI environment. The viability of astrocytes was measured by CCK-8, and the cytoskeleton changes were observed by Phalloidin- TRITC staining. The expressions of differentiation markers, Cx43 and phosphorylated Cx43 (P-Cx43) of A1/A2 astrocytes were detected by Western blot, and the complement C3 and S100A10 of A1/A2 astrocytes were detected by ELISA. The TBI rat model was established. The water content of brain tissue was measured by dry-wet specific gravity method, the pathological morphology of brain tissue in cortical injury area was observed by HE staining method, ROS was detected by fluorescence quantitative method, Cx43 expression was detected by immunohistochemistry method, and the differentiation markers of A1/A2 astrocytes were detected by immunofluorescence. ResultsIn the TBI environment, astrocytes showed decreased cell viability, blurred skeleton, and increased expression of Cx43. In TBI rats, the water content of brain tissue increased, the brain tissue in the cortex injury area was seriously damaged, ROS and Cx43 expression were significantly increased, and mainly distributed in A2 astrocytes. Remazolam can reverse the above results after the intervention. ConclusionRemazolam affects the phenotype and function of astrocytes to improve TBI via regulating Cx43, and plays a role in protecting the neurological function of TBI rats.

Pharmacokinetics, tissue distribution, and subacute toxicity of oral carrageenan in mice

Carrageenan (CGN) is a typical sulfated polysaccharide widely applied in the food and pharmaceutical industries. Its in vivo behavior plays vital roles in understanding structural and biological functional relationships. The lack of UV chromophores in highly sulfated polysaccharides presents a challenge for their in vivo behavior studies. Therefore, this study aimed to develop a fast and effective quantitative fluorescence method for investigating the pharmacokinetics and tissue distribution of CGN. Fluorescence isothiocyanate labeling of CGN (FCGN) and microplate reader-based measurements were developed and validated to study its pharmacokinetics. These results showed that the FCGN concentration peaked at 3 h, the mean residence time was 36.6 h, and the clearance rate was 0.1 L/h/kg. Most of the FCGN was excreted in the feces, while 9.2 % was excreted in the urine, suggesting absorption and metabolism. The pharmacokinetic parameters indicated that the FCGN was absorbed quickly, eliminated slowly, and could remain in the body for a sustained profile. Moreover, ex vivo imaging and quantification of FCGN in tissues revealed that FCGN accumulated in the liver and kidney. Furthermore, oral administration of CGN or KOs for 14 days led to changes in liver and kidney indices. Histological analysis of significant organs revealed hepatocyte necrosis in the liver, renal tubular vacuolization in the kidney, and incomplete colonic epithelial cells. The KOs had a more significant effect on inflammatory cell infiltration than did CGNs. These in vivo findings laid the foundation for the study and application of CGN in food and pharmaceutical applications.

Post delivery withdrawal time of tenofovir for blocking mother to child transmission of HBV in women with high HBV load during late pregnancy

Purpose: To study the withdrawal time of tenofovir disoproxil fumarate (TDF) after delivery in parturient women having high hepatitis B virus (HBV) load who took TDF during late pregnancy to block fetal contact with HBV.
 Methods: This is a retrospective analysis of clinical data of 77 pregnant women close to parturition with high HBV density in Southern Central Hospital of Yunan Province from February 2020 to February 2021. Based on time of drug discontinuation, they were assigned to group A (immediately after delivery, n = 24); group B (1 month after delivery, n = 26), and group C (3 months after delivery, n = 27). All three groups took TDF before delivery. Liver function, with alanine aminotransferase (ALT) was assessed using Roche detection system. Deoxyribonucleic acid of hepatitis B virus (HBV-DNA) was assayed by fluorescence quantitative method. Hepatitis B surface antigen (HBsAg) and HB e antigen (HBeAg) were measured using chemiluminescence microparticle immunoassay (CMIA). All assays were done after 2, 4, 8, 12, 18 and 24 weeks of drug withdrawal.
 Results: All parameters in the three groups of patients returned to normal levels after 2, 4, 8, 12, 18 and 24 weeks of drug withdrawal. In 83 neonates, tests for hepatitis B DNA and surface antigen were negative 1 month after birth, with 100 % block of maternal neonatal transmission, and no HBV infection. The incidence of adverse reactions in pregnant women were 16.67, 19.24 and 18.52 % in groups A, B and C, respectively, with no significant difference (p > 0.05).
 Conclusion: The therapeutic effect of TDF for parturient women with high HBV density is not related to drug withdrawal time after delivery. It is necessary to increase the number of samples in subsequent studies in order to validate the research results.

Screening results and mutation frequency analysis of G6PD deficiency in 1,291,274 newborns in Huizhou, China: a twenty-year experience.

This study aimed to investigate the incidence rate and spectrum of gene mutations of Glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Huizhou city of southern China to provide a scientific basis for disease prevention and control in the area. From March 2003 to December 2022, newborn screening for G6PD enzyme activity was carried out in Huizhou city using the fluorescence quantitative method. Infants who tested positive during the initial screening were diagnosed using the nitroblue tetrazolium ratio method, while a subset of infants received further gene mutation analysis using the multicolor probe melting curve analysis method. A total of 1,291,274 newborns were screened and the screening rate has increased from 20.39% to almost 100%. In the 20-year period, 57,217 (4.43%) infants testing positive during the initial screening. Out of these infants, 49,779 (87%) were recalled for confirmatory testing. G6PD deficiency was confirmed in 39,261 of the recalled infants, indicating a positive predictive value of 78.87%. The estimated incidence rate of G6PD deficiency in the region was 3.49%, which was significantly higher than the average incidence rate of 2.1% in southern China. On the other hand, seven pathogenic G6PD variants were identified in the analysis of the 99 diagnosed infants with the most common being c.1388 G > A (48.5%), followed by c.95 A > G (19.2%), c.1376 G > T (15.2%), c.871 G > A (9.1%), c.1360 C > T (3.0%), c.392 G > T (3.0%), and c.487 G > A (1.0%). The incidence of G6PD deficiency in newborns in the Huizhou city was higher than the southern China average level, while the types and frequencies of gene mutations were found to vary slightly from other regions. Our findings suggested that free government screening and nearby diagnosis strategies could reduce the incidence of G6PD deficiency in the area.

Establishment of SYBR Green I Real-Time PCR for Detection of Streptococcus agalactiae in Aquaculture Waters

Streptococcus agalactiae has a serious negative impact on tilapia aquaculture, and rapid detection of trace S. agalactiae in aquaculture waters and timely and effective sterilization measures could significantly reduce the probability of its outbreak in tilapia farming. Here, we established a fluorescence quantitative detection method of S. agalactiae in tilapia aquaculture waters based on the Streptococcus agalactiae CAMP factor (cfb) (GU217532.1) sequence of its CAMP factor. The results showed that the Ct value and logarithm of plasmid copy number presented a good linear relationship (y = −3.49x + 38.78; R2 = 0.997) in the plasmid concentration range of 1.57 × 102−1.57 × 109 copies/μL, and the lowest concentration for sensitive detection of the established method was 1.57 × 102 copies/μL. The method exhibited high specificity for the detection of S. agalactiae and generated negative results when using the DNA of Aeromonas veronii, Staphylococcus epidermidis, Aeromonas hydrophila, and Edwardsiella tarda as templates for qPCR. Intra- and inter-group repeated experiments produced variation coefficients lower than 2%, indicating high stability and specificity of the method. The method was then used to detect S. agalactiae in two tilapia aquaculture waters. The results showed that the concentration of S. agalactiae was 12 copies/mL and 2 copies/mL in the two water samples, respectively. The method can directly detect trace S. agalactiae in the aquaculture water and facilitate the diagnosis, prevention, and control of S. agalactiae in tilapia aquaculture, which will greatly reduce the probability of large-scale outbreak of S. agalactiae and economic loss.

The Effect of Rootstock on the Activity of Key Enzymes in Acid Metabolism and the Expression of Related Genes in ‘Cabernet Sauvignon’ Grapes

The types and contents of organic acids in wine grapes determine wine quality. To explore the effects of different rootstocks on the acid metabolism of ‘Cabernet Sauvignon’ grapes, various perennial rootstock–scion combinations were used as experimental materials. High-performance liquid chromatography (HPLC) was used to determine citric acid, tartaric acid, and malic acid contents during fruit development. Succinic acid and oxalic acid contents and the activity of related enzymes were measured using spectrophotometry. The expression levels of related genes were measured using a real-time fluorescence quantitative method. The results showed that all four rootstock types significantly reduced oxalic acid and citric acid contents in the grapes, while increasing succinic acid content to varying degrees. Employing 110R, SO4, and Kangzhen3 rootstocks increased tartaric acid and malic acid contents. Enzyme activity analysis revealed that 110R, SO4, and Kangzhen3 rootstocks increased the NAD-MDH enzyme activity, which positively correlated with malic acid content. Simultaneously, these rootstocks reduced the NADP-ME enzyme activity level. NAD-MDH and PEPC gene expression levels were higher in ‘Cabernet Sauvignon’ grapes grafted with 110R, SO4, and Kangan3 rootstocks compared to control self-rooted seedlings. Grafting these three rootstocks enhanced malic acid accumulation in ‘Cabernet Sauvignon’ grapes.

The bio-derived material acacetin ameliorated hyperlipidemia and intestinal barrier damage in mice by modulating gut microbiota

The increasing incidence of hyperlipidemia has been linked to disruptions in gut flora. The bio-derived material, acacetin, is a naturally flavonoid compound extracted from various plants that has been shown to protect the liver and lowers blood lipid levels, its potential to mitigate gut barrier damage caused by a high-fat diet (HFD) has not been fully explored. This work aimed to investigate the effects of acacetin on HFD-induced intestinal barrier disruption and its potential interaction with gut microbiota regulation. Forty-eight female ICR mice were divided into three groups: control group (standard diet containing 6% daily energy from fat), HFD group (45% of daily energy from fat), and the acacetin (AC) group (HFD with acacetin 30 mg/kg body weight). The test period lasted for eight weeks. In addition to lipid metabolism parameters, serum lactate dehydrogenase (LDH) activity, diamine oxidase (DAO) activity, and lipid metabolism, we used the real-time fluorescence quantitative method to measure the expression of zonula occludens 1 (ZO-1) and occludin genes. The microorganisms were analyzed by 16 s RNA and functional gene prediction analysis. The results indicated that acacetin treatment could alter serum biochemical parameters and reduce body weight, liver weight gain, and abdominal fat accumulation. Furthermore, acacetin increased the expression levels of ZO-1 and occludin in HFD mice. In addition, acacetin altered the structure, diversity, and function of intestinal flora, characterized by the restoration of the Firmicutes/Bacteroidetes ratio. Additionally, the species abundance were significant correlation with lipid factors, DAO, and LDH. Alistipes and Acetatifactor were the prevalent genus in the AC group. Acacetin downregulated HFD-induced Facultative_anaerobic phenotypes related to Clostridium according to the BugBase analysis. The KEGG study revealed that acacetin altered the functional composition of microorganisms, as evidenced primarily by variations in the abundance of metabolic pathways involved in lipid metabolism and intestinal epithelial injury. The COG category showed acacetin increased the abundance of Cytoskeleton associated with the intestinal barrier. Overall, acacetin ameliorated HFD-induced hyperlipidemia and intestinal barrier damage in mice by modulating intestinal bacteria, exhibited a good clinical application prospect.

Topical fluoride treatment effectiveness: a systematic review

Relevance. The systematic review aimed to evaluate the effectiveness of topical fluoride in children based on the analysis of modern randomized clinical trial (RCT) data.Materials and methods. This review was carried out using the PRISMA checklist (the Preferred Reporting Items for Systematic Reviews and Meta-Analyses). The proposed criteria determined the quality of the studies for inclusion in the review and then during the result summary. A preliminary search was conducted among articles published between 2013 and 2023 where fluoride varnishes were one of the preventive products. The search included MeSH terms, the keywords "fluorides", "fluorine varnish", "caries prevention", "remineralisation", and "children". A preliminary search found 346 studies. After excluding duplicates and irrelevant studies, two independent reviewers analysed nine RCTs based on the PRISMA criteria.Results. Children aged eight months to five years old participated in five out of nine RCTs on the fluoride varnish application included in the review, and four studies included adolescents aged 14–20 y.o. The duration of the studies ranged from 2 months to 3.5 years. Six studies described the use of fluoride varnish in children, and three studies reported on the application of calcium phosphate gels with fluoride versus fluoride varnish. Various ways determined enamel condition, namely, visual method, digitally processed photography, analysis of radiographs, QLF (quantitative light fluorescence method), and laser fluorescence analysis method (Diagnodent). The dmft, ICDAS indices, caries increment, the WSL (white spot lesion) index, and the assessment of changes in the tooth damage area evaluated the prevalence and intensity of caries. In the control groups, most studies used 1000 to 1500 ppm fluoride toothpastes according to the usual scheme. None of the studies statistically proved the additional preventive effectiveness of the fluoride varnishes.Conclusions. The analysis of RCTs did not reveal the advantages of fluoride varnishes for caries prevention in children and adolescents compared to standard oral hygiene procedures using fluoride toothpastes.

Quantification of lateral associations of achondroplasia in FGFR3.

Fibroblast growth factor receptor 3 (FGFR3) belongs to the receptor tyrosine kinase (RTK) family and is a key player in bone growth, tumor angiogenesis, and other developmental processes. Of particular interest is a substitution of glycine to arginine (G380R) in the transmembrane domain, which is implicated in human achondroplasia. The G380R mutation has been reported to stabilize FGFR3 dimers in the absence of ligand; however, recent work has suggested that the G380R mutation may be inducing the formation of FGFR3 oligomers that are larger than dimers. This has propelled us to re-evaluate the effect of the mutation on FGFR3 self-association in the plasma membrane of live cells using a quantitative fluorescence method, FSI-FRET, which reports on oligomer size, stability, and structure. This project aims to re-examine the self-association of both wild type and G380R mutant FGFR3 over physiologically relevant receptor concentrations and under different growth factor conditions using FSI-FRET.

Shikonin promotes hypertrophic scar repair by autophagy of hypertrophic scar-derived fibroblasts.

To investigate the Shikonin (SHI) induce autophagy of hypertrophic scar-derived fibroblasts (HSFs) and the mechanism of which in repairing hypertrophic scar. This study showed that SHI induced autophagy from HSFs and repaired skin scars through the AMPK/mTOR pathway. Alamar Blue and Sirius red were used to identify cell activity and collagen. Electron microscopy, label-free quantitative proteomic analysis, fluorescence and other methods were used to identify autophagy. The differences in the expression of autophagy and AMPK/mTOR pathway-related proteins after SHI treatment were quantitatively analyzed by Western blots. A quantitative real-time polymerase chain reaction assay was used to detect the expression of LC3, AMPK and ULK after adding chloroquine (CQ) autophagy inhibitor. After treatment with SHI for 24 hours, it was found that the viability of HSFs was significantly reduced, the protein expression of LC3-II/LC3-I and Beclin1 increased, while the protein expression of P62 decreased. The expression of phosphorylated AMPK increased and expression of phosphorylated mTOR decreased. After the use of CQ, the cell autophagy caused by SHI was blocked. The key genes LC3 and P62 were then reexamined by immunohistochemistry using a porcine full-thickness burn hypertrophic scar model, and the results verified that SHI could induce autophagy in vivo. These findings suggested that SHI promoted autophagy of HSFs cells, and the potential mechanism may be related to the AMPK/mTOR signal pathway, which provided new insights for the treatment of hypertrophic scars.

The Protective Effect and Mechanism of Baicalin on Postoperative Cognitive Function and Nerves in Aged Rats

To explore the effect of baicalin on cognitive function and nerves after splenectomy in aged rats is the main objective of the study. Aged rats were randomly divided into a sham operation group, a model group (to construct a splenectomy surgical model) and a baicalin treatment group (baicalin was given after the model was constructed). The pathological conditions like interleukin-6, tumor necrosis factor alpha, interleukin-1 beta content; brain detection by terminal deoxynucleotidyl transferase dUTP nick end labeling method apoptosis of tissue cell; western blot method was used to detect the expression of related proteins and real-time fluorescence quantitative polymerase chain reaction method was used to detect the messenger ribonucleic acid levels of related genes. Compared with the control group, the brain tissue cell apoptosis increased and the expression of inflammatory factors increased in the model group; compared with the model group, the brain tissue apoptosis of the baicalin treatment group decreased, the expression of Ki67 positive cells increased and the inflammation-related factor interleukin-6, tumor necrosis factor alpha, interleukin-1 beta levels were significantly decreased, N-methyl D-aspartate receptor subtype 2B, phosphorylated-extracellular signal-regulated kinase, phosphorylated-cAMP response element-binding protein expressions were significantly increased. Baicalin can improve postoperative cognitive dysfunction and nerve injury in aged rats after splenectomy by improving the pathological state of brain tissue, attenuating the inflammatory response and promoting apoptosis of brain tissue.

Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR

The natural host range for brassica yellows virus (BrYV) is generally limited to Cruciferae. However, we found that BrYV can naturally infect strawberry. The full-length genome sequences of BrYV-MB (accession No. MZ666129) and BrYV-HY (accession No. ON060762) identified in strawberry from Yantai and Beijing, China, were obtained by high-throughput sequencing (HTS) combined with the RT-PCR and RACE techniques. The complete genome sequences of BrYV-MB and BrYV-HY are 5666 nt and contain six open reading frames (ORFs). The two isolates have the highest nucleotide (nt) sequence identity of 99.0%. The infectious cDNA clone of BrYV-HY was constructed through homologous recombination and used to agroinfiltrate Nicotiana benthamiana and Arabidopsis thaliana. The inoculated leaves of N. benthamiana showed necrotic symptoms after 4 days of inoculation (dpi), and the systematic leaves of A. thaliana exhibited purple symptoms at 14 dpi. To develop a rapid and high-sensitive method for the detection of BrYV, a TaqMan real-time fluorescence quantitative RT-PCR method (TaqMan RT-qPCR) was established. Under optimum reaction conditions, the sensitivity of the detection was as low as 100 fg and approximately 100-fold more sensitive than the conventional RT-PCR, so it can be used in large-scale testing.

A Quantitative Fluorescent Marker Method for Evaluation of Surface Cleanliness Using Digital Image Data

医療環境の清掃を評価する方法の一つである蛍光マーカー法は安価で簡便でリアルタイムに清掃状態を可視化できるが評価は定性的で微生物除去との関連性も十分検討されていない．そこで今回写真画像に着目し蛍光マーカー（Fm）を撮影した画像のピクセル値（PX）を用いた定量法の構築と微生物（Bc）除去率との関連性を検討した．まず「ピクセル上のFm量の増加に従いPX値は最大PX値（255）に対して指数関数的に漸近する」との仮説式により総Fm量（Xt）を算出した．次に市販のFmの希釈液の一定量を塩化ビニール黒，白，ステンレス（S）の3種の基板に塗布しデジタルカメラで撮影してその各画像のPXからXtを算出し実塗布量との相関係数Rから仮説式を検証した．Bc除去率にはStaphylococcus aureusを使用した．白，Sの各板上にBcおよびFmを隣接塗布し含水ワイプで一方向にふき取りその後のFmとBcの各除去率を算出した．結果，仮説式に基づき算出したXtと実塗布量は良好な相関性（黒，白，S板上での相関係数は各々0.99，0.99，0.99）を示した．また白，S板上のBc，Fmの各除去率の決定係数は各々0.46，0.61であった．一方Fm除去率90％以上に限ればBc除去率との一致性は良好な傾向を示した．これらよりFm量を画像データから定量化することは可能であることが検証された．

The crosstalk fluorescence spectroscopy analysis principle and an accurate fluorescence quantitative method for multi-composition fluorescence substances

Fluorescence quantitative analysis methods are extensively used in biomedicine inspection, petrochemical industry, environmental monitoring, and many other fields in the past decades. When the analyte is composed of multiple compositions, the accuracy of the conventional method declines significantly due to the fluorescence spectral crosstalk. In this research, the interactions between the light and the multiple compositions are comprehensively analyzed. The concepts of the quenching due to mutual absorption and the fluorescence overlapping are considered, and the mechanism of multi-composition fluorescence emission under single-wavelength excitation light is analyzed theoretically. The mixture experiment and the dilution experiment are designed to illustrate that the quenching due to mutual absorption has a significant nonlinear impact on fluorescence quantitative analysis and the mechanism of fluorescence spectral crosstalk gives a good explanation for these experiments. Through the in-depth theoretical analysis, the computer simulation, and the experiments, a novel principle named the Crosstalk Fluorescence Spectroscopy Analysis (CFSA) is proposed and verified, which has much higher quantitative analysis accuracy (R2>0.99 and RMSE≤0.2) than the conventional methods when analyzing the multi-composition samples. Unlike many correction approaches to fluorescence spectroscopy, the novel CFSA can serve as a complete analysis method rather than a correction method. These concepts and the principle are expected to be applied in many practical analysis fields.

Effects of exosomes from human adipose-derived mesenchymal stem cells on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice

Effects of exosomes from human adipose-derived mesenchymal stem cells on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice

EV71 was Detected by Real-Time Fluorescence Quantitative RT-PCR Combined with Hand-Foot-Mouth Disease Rhesus Monkey Model

In this study, a real-time fluorescence quantitative RT-PCR method was developed for the detection of enterovirus 71 (EV71) and the detection of EV71 viral load during the establishment of hand-foot-mouth disease (HFMD) model in rhesus monkeys. The model of hand-foot-mouth macaque was established by injecting newborn macaques infected with the virus venom, and the dynamic changes of virus content in blood samples from 0 to 14 days after infection were monitored by real-time quantitative RT-PCR. The linear range of the standard curve was between 103 and 108 copies/µL, and the PCR amplification rate was 99.37%. The results showed that the established method could detect a minimum of 102 copies of viral RNA, 100 times higher than the conventional RT-PCR method. It has the characteristics of strong specificity, high sensitivity and good stability. The results of blood sample analysis showed that the three groups of rhesus monkey models were successfully infected with EV71, and the copy number of virus proliferation was correlated with time and challenge dose. The results showed that real-time quantitative PCR could be used for quantitative detection of EV71 viral load.

A Loop-mediated Isothermal Amplification With a Nanoparticle-Based Lateral Flow Biosensor Assay to Detect Pseudomonas aeruginosa in Endophthalmitis.

Purpose Pseudomonas aeruginosa is the most common bacteria causing endophthalmitis after cataract surgery. Vitreous fluid culture and molecular studies are commonly used in clinical diagnoses, but have disadvantages, such as a long culture cycle and low detection sensitivity. Here, we report a loop-mediated isothermal amplification (LAMP) method combined with the nanoparticles-lateral flow biosensor (LFB) method for rapid and specific detection of P. aeruginosa.MethodsA set of six primers was designed to target the OprL gene of P. aeruginosa. Genomic DNA extracted from several gram-negative and gram-positive bacteria was used to determine the sensitivity and specificity of the analysis. LAMP reactions were conducted at 65 °C for 50 minutes, and results were reported using the LFB method.ResultsThe DNA template of P. aeruginosa was specifically recognized by the P. aeruginosa-LAMP-LFB (PA-LAMP-LFB) method as no cross reactions were observed for non–P. aeruginosa templates. The analytical sensitivity of our assay was 100 fg per test for the pure cultured DNA template, and the result obtained using the LFB was consistent with that of colorimetric indicator detection. The whole test could be completed within 1h. This method was used to detect P. aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae; only P. aeruginosa was positive. The positive rates of P. aeruginosa detected by a traditional culture method, the LAMP-LFB method, and the fluorescence quantitative polymerase chain reaction method were 17.7%, 17.7%, and 13.3%, respectively.ConclusionsThe P. aeruginosa-LAMP-LFB method established here is a rapid, specific, and sensitive method for the detection of P. aeruginosa, which can be widely used.

The Therapeutic Effects of Oral Intake of Hydrogen Rich Water on Cutaneous Wound Healing in Dogs.

This study explored the effects of drinking Hydrogen-rich water (HRW) on skin wound healing in dogs. Eight circular wounds were analyzed in each dog. The experimental group was treated with HRW thrice daily, while the control group was provided with distilled water (DW). The wound tissues of dogs were examined histopathologically. The fibroblasts, inflammatory cell infiltration, the average number of new blood vessels, and the level of malondialdehyde (MDA) and superoxide dismutase (SOD) activity in the skin homogenate of the wound was measured using the corresponding kits. The expressions of Nrf-2, HO-1, NQO-1, VEGF, and PDGF were measured using the real-time fluorescence quantitative method. We observed that HRW wounds showed an increased rate of wound healing, and a faster average healing time compared with DW. Histopathology showed that in the HRW group, the average thickness of the epidermis was significantly lower than the DW group. The average number of blood vessels in the HRW group was higher than the DW group. The MDA levels were higher in the DW group than in the HRW group, but the SOD levels were higher in the HRW group than in the DW group. The results of qRT-PCR showed that the expression of each gene was significantly different between the two groups. HRW treatment promoted skin wound healing in dogs, accelerated wound epithelization, reduced inflammatory reaction, stimulated the expression of cytokines related to wound healing, and shortened wound healing time.

Feasibility study of fluorescence quantitative PCR for the detection of microecological dynamics in fermented maize stover feeds

During the fermentation of corn stalk bio-feed, the quantity of bacteria in corn stalk bio-feed was counted by fluorescence quantitative PCR and plate colony counting respectively. The comparative analysis of these studies was used to explore the feasibility of fluorescence quantification methods and changes in microbiota during fermentation. The results showed that the standard deviation of fluorescence quantitative method was smaller than that of plate method, but the trend was similar. The biomass of Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae reached their maximum on the third, fifth and fifth day respectively, and then decreased gradually and maintained at a certain level. The experiment showed that the fluorescence quantitative PCR method can accurately quantify the number of bacteria in corn stalk bio-feed, and it is a better method to quantitatively detect the dynamic changes of different kinds of bacteria in corn stalk bio-feed.