Establishment and verification of a real-time quantitative PCR method for determination of exogenous antibody gene copy number in CHO cells / 中国生物制品学杂志

@#Objective To establish a real-time quantitative PCR method using SYBR GreenⅠto detect the copy numbers of light chain(LC)and heavy chain(HC)of exogenous antibody gene in CHO cells,and verify and preliminarily apply this method.Methods With the B2m(β2-microglobulin)expressed stably in CHO cells as the internal reference gene,suitable primers of LC,HC genes and internal reference gene were designed respectively,and the reaction system and program of the real-time quantitative PCR method were determined. The established method was verified for the specificity,linearity,precision and durability,and used to detect the copy numbers of LC and HC genes in the recombinant cell lines of working cell bank(WCB)and cells of different passages.Results The primers of exogenous genes and internal reference gene showed specific binding to the target fragments;The efficiency of primer amplification for the B2m gene,LC gene,and HC gene was 106. 7%,106. 3% and 99. 1%,respectively,and the correlation coefficients of the linear equations were all greater than 0. 99 with a good linear relationship;The relative standard deviations(RSDs)of precision verification were all less than 1%;Few cycles of freeze-thaw in a short period had little effect on the detection results. The copy numbers of LC and HC genes in different generations of recombinant cell lines detected by the established method showed no obvious changes.Conclusion A real-time quantitative PCR method for the determination of the copy number of exogenous genes in CHO cells was successfully established with good specificity,linearity,precision and durability,which provides a reference for detecting the copy number of exogenous genes expressed in other CHO cell lines

Detecting the copy number of orfA gene to identify Brucella species and biovars by a real-time quantitative PCR assay / 中华地方病学杂志

Objective:To establish a real-time quantitative PCR assay to detect the copy number of IS711 transposase gene (orfA) in Brucella genome, and the assay is applied to identify the species and biovars of Brucella. Methods:To establish an orfA gene copy number detection system based on Taqman real-time quantitative PCR technique. Primers and probes of bcsp31 and orfA genes were designed, the contents of bcsp31 gene and orfA gene in the same strain with the same DNA concentration were simultaneously detected by real-time quantitative PCR assay, and cycle number (CT value) of the two genes were obtained. According to the differences of CT values of bcsp31 gene and orfA gene, the copy number of orfA gene in Brucella genome was calculated. At the same time, the DNA of Brucella 16M strain was double decreasing dilution to verify the stability of the detection system. Results:A real-time quantitative PCR assay was used to detect bcsp31 gene and orfA gene simultaneously, when the DNA concentration difference of 16M strain was 2 times, the mean difference of CT values measured was 1.00, 95% confidence interval was 0.95-1.05, standard deviation was 0.17, and coefficient of variation was 0.17. The orfA gene copy number of 30 Brucella strains was detected by this detection system. It was found that there were 6, 9, and 7 copy numbers in the biovars 1-3 of Brucella melitensis, respectively. The strain of Brucella suis biovar 2 had 10 copy numbers, which were different from those of the other 4 strains of biovars 1, 3-5. There were 37 copy numbers in Brucella ovis strain. The copy numbers were stable at 5-6 copies in 8 biovars (1-7, 9) of Brucella abortus strains. Conclusions:A real-time quantitative PCR assay for detection of orfA gene copy number in Brucella DNA has been established. This method could identify some Brucella species and biovars strains.

Reference gene screening for Real-time quantitative PCR in Polygonum multiflorum / 中国中药杂志

To select suitable references gene of Polygonum multiflorum for gene expression analysis in different tissues, five candidate reference genes like Actin,GAPDH,SAND,PP2A,TIP41 were selected from the transcriptome data of P. multiflorum, then the specific primers were designed. The expression stability of the five reference genes in different tissues of P. multiflorum was analyzed by Real-time quantitative PCR through avilable analysis methods such as geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, to ensure the reliability of the analysis results. The results showed that there were significant differences in the expression levels and stability of candidate genes in different tissues of P. multiflorum. Ct distribution analysis of the expression levels of candidate genes showed that the expression levels of Actin and GAPDH genes were relatively high in different tissues, while the expression levels of SAND, PP2A and TIP41 were lower. The stability of each candidate gene was analyzed by different methods. The results of geNorm analysis showed that the expression of PP2A and GAPDH was the most stable, the expression stability of SAND was the worst, the stability of PP2A was the highest in both NormFinder and Delta CT, the stability of SAND was the lowest, and the stability of Actin was the most stable in BestKeeper analysis. Through the comprehensive evaluation and analysis of the stability of candidate genes by RefFinder, it is concluded that the stability of PP2A gene is the highest, followed by GAPDH, Actin, TIP41, SAND, and SAND gene is the worst. Therefore, the PP2A gene is an ideal reference gene for the analysis of gene expression in different tissues of P. multiflorum.

Detection of measles virus infection and proliferation by real-time quantitative PCR / 上海预防医学

Objective A SYBR-Green real-time quantitative PCR(RT-qPCR)method was set up to detect the infection and proliferation of measles virus, which could be useful in virus titer determination. Methods In this project, we used a 405 bp fragment of the N gene of measles virus as a target sequence and constructed a plasmid to establish the standard curve in absolute quantitative experiment. We then used this method to obtain the proliferation curve of measles virus and to detect the virus proliferation at different MOI. Results There was a linear relationship between the virus copy number and the titer of the measles virus reference at the range of 6 to 2 lgCCID50/mL, with a correlation coefficient (r) of 0.991(P < 0.01). Based on the analysis of virus proliferation curve, measles virus mainly proliferated intracellularly within 48 h after its entering the cell. There was no detected increase in viral RNA level in the first 24 h, suggesting the virus was in a silent period in the cell. After 24 h, the virus expanded in large numbers and entered the exponential growth phase. The intracellular viral RNA level reached the plateau phase after its peak at 96 h. The virus secreted to the outside of the cell entered the exponential growth phase starting from 48 h, peaked at 144 h, then followed by plateau phase. Conclusion A SYBR-Green RT-qPCR method is established and used to monitor virus proliferation. Our result is helpful in understanding of the proliferation and secretion of measles virus in cells and provides experimental basis for detection of live attenuated virus titers.

Selection of reference genes for quantitative real-time PCR in Morinda officinalis / 中草药

Objective: To select the appropriate reference genes for calibrating the quantitative real-time PCR detection of gene expression in different tissues and leaves with different treatments of Morinda officinalis. Methods: With different groups and different processing leaves of M. officinalis as materials, 10 internal genes, including GAPDH, CYP, TUA, Actin and so on, were selected as candidate genes according to the M. officinalis transcriptome data. The expression stability of internal reference genes was analyzed by using real-time fluorescence quantification technique combined with software such as geNorm, NormFinder and BestKeeper, so as to select stable reference genes in different tissues and leaves of M. officinalis with different treatments. Finally, appropriate internal reference genes were selected to analyze the relative expression levels of DXS and DXR genes in different tissues and leaves with different treatments. Results: Internal reference genes GAPDH and UBQ were the most stable in different tissues of M. officinalis, the double internal reference combination of GAPDH + UBQ can more accurately analyze the relative expression levels of target genes in different tissues of M. officinalis, while the most stable reference genes in leaves with different treatments were GAPDH and Actin; The selection of the double reference combination of GAPDH + Actin can ensure the reliability of the target gene expression results. In different tissues of M. officinalis, the relative expression of DXS target gene was in sequence of root < stem < leaf, while the relative expression of DXR was stem < root < leaf. The relative expression levels of DXS and DXR genes in leaves with different treatments were increased compared with those untreated leaves (CK). Conclusion: The selected stable internal reference genes lay a foundation for the subsequent study on the expression of related genes of M. officinalis. Using the combination of two stable internal references to homogenize the target genes is conducive to improving the accuracy of the analysis of the expression of target genes.

Optimization of a fluorescent qPCR detection for RNA of SARS-CoV-2 / 生物工程学报

We optimized a fluorescent quantitative polymerase chain reaction (qPCR) assay system for rapid and real time detection of SARS-CoV-2 RNA. The results show that the lowest dilution of RNA samples used for the detection of SARS-CoV-2 RNA could reach 1/10 000 (the initial value is set as 10 ng/μL). Moreover, the cycle threshold (Ct) for samples of clinically diagnosed COVID-19 was lower than 35 or 40. The sensitivity of this method was satisfactory. The results were consistent with those of the COVID-19 detection kit on the market under the same conditions, but the number of cycles required was shortened by about 2. Therefore, the optimized assay developed in this study can be used in screening and early clinical diagnosis. Our work provides a tool to facilitate rapid clinical diagnosis of COVID-19.

Selection and validation of internal reference genes for qPCR in Polygonatum cyrtonema tubers at different development stages and in response to abiotic stress / 中国中药杂志

In order to analyze the expression of genes involved in steroidal saponin biosynthesis pathway in Polygonatum cyrtonema tubers, it is very important to select internal reference genes that are stably expressed at different development stages and in response to abiotic stress. According to the previously established P. cyrtonema transcriptome database and reported internal reference genes in plant, this study systematically analyzed eight candidate internal reference genes including histone H2 A, glyceraldehyde-3-phosphate dehydrogenase, ACTIN, β-tubulin, ubiquitin-conjugating enzyme-E2-10, elongation factor 1-alpha isoform, 18 S rRNA and α-tubulin 4 for expression stability in P. cyrtonema tubers at different development stages and in response to methyl jasmonate(MeJA) stress by using Real time fluorescence quantitative PCR(qPCR). Based on the statistical analysis of qPCR results by using GeNorm, NormFinder and BestKeeper softwares, the expression of ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform are the most stable in P. cyrtonema tubes at different development stages and in response to MeJA stress. The two internal reference genes were further validated by analyzing the expression of 4 genes involved in steroidal saponin biosynthesis pathways. In conclusion, ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform can be used as the most appropriate internal reference genes for qPCR analysis in P. cyrtonema. This study also provide a foundation for future investigate the molecular mechanism of steroidal saponin biosynthesis pathways in P. cyrtonema.

Identification of stable reference gene by qRT-PCR in Senecio scandens / 中国中药杂志

As a traditional Chinese medicine, Senecio scandens is rich in important compounds such as flavonoid and sesquiterpenoid. Based on the transcriptome data of S. scandens, 15 candidate reference genes were selected including ABCT, ACT1, ACT2, ACT3, ACBP, ARF, ATPS, EF-H, EF-1α, ETIF, GAPDH, GTPB, MPS, UCE and 60S. Firstly, 9 candidate genes with relatively stable expressions such as ACT1, ACBP, ARF, ATPS, EF-1α, GAPDH, MPS, UCE and 60S were screened from different tissues of S. scandens by RT-PCR. Then, qRT-PCR was used to quantitatively analyze gene expression of these nine candidates in S. scandens with or without stress treatments. Further analysis of these gene expression data by geNorm and NormFinder showed that ACT1 exhibited the stablest expression in all samples and could serve as a reference gene for future study of S. scandens, and provide an endogenous control for gene expression analysis.

An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels / 中华血液学杂志

Objective@#To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison.@*Methods@#Peking University People’s Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated.@*Results@#①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories’ results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH.@*Conclusion@#The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

Effect of endophytic fungi on expression of key enzyme genes in polysaccharide and alkaloid synthesis from Dendrobium officinale / 中草药

Objective: To analyze the effect of endophytic fungi GXRz2, GXRz3 and GXRz10 on content of poly saccharide and alkaloid, and the expression of key enzyme genes UGPase, HMGR, and FPS in Dendrobium officinale. Methods: The endophytic fungi liquid were added to D. officinale seedlings. Polysaccharide and alkaloid content were measured by spectrophotometry method. With 18S rRNA as internal control gene, the expression of key enzyme genes was detected by real-time quantitative PCR method. Results: It was found that the content of polysaccharide in D. officinale was higher, mainly concentrated in stem, but the lowest in root. And the content of alkaloid in D. officinale was lower, mainly accumulated in leaf, but the lowest in root. In addition, the three endophytic fungi strains could promote accumulation of polysaccharide and alkaloid in D. officinale to a certain extent. The expression of UGPase, HMGR and FPS genes in D. officinale induced by different strains was detected by real-time quantitative PCR. The results showed that endophytic fungi GXRz3 and GXRz10 could significantly increase the expression of UGPase, HMGR and FPS genes in D. officinale. In the polysaccharide synthesis pathway, the UGPase gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. In the alkaloid synthesis pathway, HMGR gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. However, FPS gene had the highest relative expression in the leaf, followed by the stem, and the least in the root. Conclusion: Endophytic fungi may affect the synthesis of polysaccharide by regulating the expression of UGPase. Considering the accumulation of polysaccharide, it is speculated that UGPase may be a key enzyme in the polysaccharide synthesis pathway of D. officinale. Endophytic fungi may affect the synthesis of alkaloid by regulating the expression of HMGR and FPS. Considering the accumulation of alkaloid, it is speculated that FPS may be a key enzyme in the alkaloid synthesis pathway of D. officinale.

Expression and clinical significance of hsa-miRNA-200c in patients with liver metastasis of epithelial ovarian cancer / 中国实用妇科与产科杂志

OBJECTIVE: To investigate the expressions of hsa-miRNA-200 c and its relationship with metastasis of EOC.METHODS: The expression of hsa-miRNA-200 c was detected by Stem-loop Real-time Quantitative PCR(TaqMan probe method)in 73 cases of EOC,30 cases of benign ovarian epithelial tumors and 30 cases of normal ovarian tissues,which were collected in gynecological operations from Guangdong General Hospital from October 2010 to May 2011.Meantime,the clinical pathologic features data were analyzed.The assessment of the correlation between hsa-miRNA-200 c and clinicopathological features,and the hierarchical analysis of hsa-miRNA-200 c level in 73 cases of ovarian epithelial cancer was further undertaken(Among 73 cases,13 patients suffered liver metastasis and 60 patients had non-liver metastasis).Overexpression or knockdown of hsa-miRNA-200 c,ovarian cancer cell invasion and migration abilitywas detected.RESULTS: The expression of miRNA-200 c in the EOC tissues was 382.18±15.22,which was significantly higher than that in the benign ovarian epithelial tumors(35.61 ± 1.42)and normal ovarian tissues(4.43 ±2.23)(P0.05).The expressions of miRNA-200 c were low in EOC with late clin-ical FIGO stage(670.91±16.88 vs. 129.52±33.3,P0.05).The transwell cabinet invasion experiment showed the expression of miRNA-200 c was negatively correlated with the invasion capability of ovarian cancer cells.CONCLUSION: MiRNA-200 c is likely to play a double regulation role in the development of EOC,whose low-expression has been associated with late EOC,lymph node metastasis,liver metastasis,and poor prognosis.

Evaluation of the transcriptional immune biomarkers in peripheral blood from warao indigenous associate with the infection by mycobacterium tuberculosis

Abstract INTRODUCTION: Biomarkers are critical tools for finding new approaches for controlling the spread of tuberculosis (TB), including for predicting the development of TB therapeutics, vaccines, and diagnostic tools. METHODS: Expression of immune biomarkers was analyzed in peripheral blood cells stimulated and non-stimulated with M. tuberculosis antigens ESAT-6, CFP10 and TB7.7. in Warao indigenous individuals. These biomarkers may be able to differentiate TB states, such as active tuberculosis (ATB) cases and latent tuberculosis infection (LTBI) from non-infected controls (NIC). A real-time reverse transcription polymerase chain reaction (RT-qPCR) assay was performed on 100 blood samples under non-stimulation or direct ex vivo conditions (NS=50) and stimulation conditions (S=50). RESULTS: The findings are shown as the median and interquartile range (IQR) of relative gene expression levels of IFN-γ, CD14, MMP9, CCR5, CCL11, CXCL9/MIG, and uPAR/PLAUR immune biomarkers. MMP9 levels were significantly higher in the LTBI-NS and LTBI-S groups compared with the NIC-NS and NIC-S groups. However, CCR5 levels were significantly lower in the LTBI-S group compared with both NIC-NS and NIC-S groups. CCL11 levels were significantly lower in the LTBI-S group compared with the NIC-NS group. CONCLUSIONS: Preliminary findings showed that MMP9 immune biomarkers separated LTBI indigenous individuals from NIC indigenous individuals, while CCR5, CCL11, CD14, and IFN-γ did not differentiate TB states from NIC. MMP9 may be useful as a potential biomarker for LTBI and new infected case detection among Warao indigenous individuals at high risk of developing the disease. It may also be used to halt the epidemic, which will require further validation in larger studies.

Utility Of Real Time Quantitative Pcr In The Diagnosis Of Congenital And Perinatal Cytomegalovirus Infection In Symptomatic Babies

Cytomegalovirus(CMV) infection may be acquired congenitally, perinatally or postnatally in babies. Congenital and perinatal CMV infection can be diagnosed by viral culture or detection and quantitation of CMV DNAby Real Time Quantitative PCR (RT-qPCR) in blood ,urine and body fluids. The objective of this study was to diagnose and determine CMV load in infants presenting with clinical features suggestive of cytomegalovirus infection by RT-qPCR of urine. This descriptive study was done on babies admitted to the Departments of Neonatology and Paediatrics Govt Medical College, Kozhikode from January 2015 to December 2017. Urine samples from 142 babies were received and processed in the Microbiology Department. DNA isolation and amplification was performed using commercial DNA extraction kit and PCR kit for detection and quantification of CMV. Serum samples of the babies with CMV viruria were tested for CMV IgM antibodies. Of 142 babies suggestive of CMVinfection CMV-DNAwas detected and quantitated in urine of 25 (17.60%) (mean age 3.36 months). . CMVIgM was positive in 15/25(60%) babies with viruria .Twenty two had congenital CMV infection (cCMV) and 3 had perinatal infection.The most common clinical presentation was jaundice 13( 52%). Of 8 babies started on Ganciclovir 7 responded to treatment. RT-qPCR helps in diagnosing and quantitating CMVload which helps in deciding on therapy and assessing response to treatment,and can predict risk for long term sequelae.

Expression of saponin biosynthesis related genes in different tissues of / 中国中药杂志

The relationship between saponin content of in different parts of the organization and expression of ginsenoside biosynthesis related gene was obtained by the correlation analysis between saponin content and gene expression. The 14 tissue parts of were studied, six saponins in Samples (ginsenoside Rg₁, Re, Rb₁, Rc, Rb₂ and Rd), group saponins and total saponins were determined by high performance liquid chromatography and vanillin-sulfuric acid colorimetric method. Simultaneously, the expression levels of 7 ginsenoside biosynthesis related genes ( and ) in different tissues of were determined by Real-time fluorescence quantitative PCR. Although 7 kinds of ginsenoside biosynthesis related enzyme gene in the involved in ginsenoside synthesis, the expression of and P450 genes had no significant effect on the content of monosodium saponins, grouping saponins and total saponins, and had significant or extremely significant on the contents of single saponins Re, Rg1, Rb1, Rd, group saponin PPD and PPT, total saponin TMS and total saponin TS (<0.05 or <0.01). The biosynthesis of partial saponins, grouping saponins and total saponins in was affected by the interaction of multiple enzyme genes in the saponin synthesis pathway, the content of saponins in different tissues of was determined by the differences in the expression of key enzymes in the biosynthetic pathway. Therefore, this study further clarified that and was the key enzyme to control the synthesis of saponins in by correlation analysis, the biosynthesis of ginsenosides in was regulated by these five kind of enzymes in cluster co-expression of interaction mode.

Study of the expressions of NF-κB and MTA2 in infiltrating ductal carcinoma of breast and their correlation / 国际检验医学杂志

Objective To investigate the expression of NF-κB and MTA2 in infiltrating ductal carcinoma of breast and their correlations to clinicopathologic character.Methods The expressions of and MTA2 in breast cancer and paired adjacent normal breast tissues of 68 breast invasive ductal cancer patients were detected by real-time quantitative PCR method,and their correlations to the clinicopathologic characteristics of breast invasive ductal cancer were analyzed.Results The expressions of NF-κB and MTA2 in breast invasive ductal carcinoma were not related to age,tumor size and histological stage (P＞0.05),and were positively correlated with lymph node metastasis and TNM stage (P＜0.05).In addition,MTA2 was highly expressed in the tissues of ER positive breast invasive ductal cancer patients (P＜0.05).There was a positive correlation between the expression of NF-κB and the expression of MTA2 in breast cancer tissue of the infiltrating ductal carcinoma of breast patients(r=0.808,P=0.012).Conclusion Measurement of NF-κB and MTA2 from breast invasive ductal carcinoma tissue may provide a theoretical basis for the diagnosis,progression and prognosis of infiltrating ductal carcinoma of breast.

Extraction and verification of miRNA from ginseng decoction / 中草药·英文版

Objective: To verify the existence of microRNAs (miRNAs) extracted from fresh ginseng decoction. Methods: Fresh ginseng was prepared into decoction according to the conventional method. The miRNA were extracted from the condensed ginseng decoction by plant microRNA extraction kit. Then miRNA were treated by DNase I and subjected to agarose gel electrophoresis and Agilent 2100 bioanalysis. MiR-159 and miR-6135, which were highly expressed in ginseng, were selected and verified by real-time quantitative PCR to detect the expression in the decoction. Results: Ginseng miRNA were successfully extracted from fresh decoction. MiR-159 and miR-6135 were expressed in fresh decoction with lower levels than those of fresh ginseng. Conclusion: miRNAs stably existed after processing, and retained some stability after high-temperature treatment. The findings provide a valuables basis for the further studies on ginseng miRNAs.

Screening of reference genes in Bupleurum scorzonerifolium and tissue expression analysis of key enzyme genes / 中草药

Objective: To select suitable references genes of Bupleurum scorzonerifolium for tissue expression analyses, and study the tissue expression characteristics of the key enzyme genes of saikosaponins biosynthesis. Methods: Five candidate reference genes including Actin, α-tubulin, β-tubulin, Cyclophilin, and EF-1α were chosen. The stability of these candidate reference genes was investigated by using four softwares (Delta CT, BestKeeper, NormFinder, and GeNorm). The stability of these candidate reference genes was tested and verified by real-time quantitative PCR. Used the stable reference gene, the tissue expression characteristics of the saikosaponins biosynthesis key enzyme genes (HMGR, IPPI, FPS, SS, and β-AS) was analyzed by qRT-PCR. Results: The average expression stability of the five candidate reference genes from high to low was β-tubulin > Cyclophilin > Actin > EF-1α > α-tubulin. Β-tubulin was the most suitable reference gene for tissue expression analysis in B. scorzonerifolium. HMGR expression level was roots > stems and fruits > leaves, IPPI expression level was roots > stems > fruits and leaves, FPS expression level was leaves > roots > stems and fruits, SS expression level was leaves > fruits > roots > stems, β-AS expression level was leaves > roots > fruits > stems. HMGR was significant positive correlated with IPPI, and FPS was significant positive correlated with β-AS (P < 0.05). Conclusion: β-tubulin gene was confirmed as the most suitable reference gene in different tissues of B. scorzonerifolium. It provided a methodological basis for the tissue expression analysis on the functional genes of B. scorzonerifolium. The expression pattern of five key enzyme genes of saikosaponins biosynthesis in different tissues had obvious differentiation, which might be involved in regulating the flow of saikosaponins synthesis and accumulation in various tissues of B. scorzonerifolium.

A real-time qPCR method to identify diatom UPA gene for the drowning diagnosis / 中国法医学杂志

Objective To establish a Real-time quantitative PCR method (qPCR) for the detection of diatom UPA barcoding genes and evaluate its application in the drowning diagnosis. Methods The homologous sequences of diatoms UPA gene was obtained by Blast from GeneBank, based on which the universal primers for diatoms were designed. DNA were extracted from 2 common human symbiotic bacteria (Escherichia coli, Bifidobacterium longum), 3 species of planktonic bacteria, 15 species of planktonic algae, tissue samples (lung, liver and kidney) from human cadavers (28 drowning victims, 1 victims by non-drowning in the water, 3 victims deaths on land) in 32 cases. The specificity, sensitivity and repeatability of the designed primers were tested. The positive rates of diatoms detection in the drowning cases were calculated. The results of the real-time quantitative method were evaluated comparatively by Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy (MD-VF-Auto SEM) and PCR-Capillary Electrophoresis (PCR-CE). Results The results showed that the primers UPA99 had strong specificity for the diatomaceae (Synedra radians, Navicula sp., Melosira varians, Cyclotella sp. and Nitzschia sp.) DNA. The melting curve of the amplified product was smooth; the peak was narrow; the melting temperature was (87±1)℃. The sensitivity of qPCR method was 1.56×10-5ng/μL with the detection range of 1.56×102ng/mL~1.56×10-5ng/μL, in contrast with the PCR-CE method (1.56×10-3ng/μL). This real-time PCR method showed high repeatability and stability with the coefficient of variation less than 2%. The detection rate of lung, liver and kidney was 89.3%, 71.4% and 64.3% respectively. Conclusion The established qPCR method, based on the universal primers designed for diatom UPA gene, has high specificity, high sensitivity and good repeatability. With a promising prospect for application, qPCR is suitable for drowning diagnosis.

Relationship between the expression level of GATA6 mRNA and the occurrence of congenital heart disease / 中华实用儿科临床杂志

Objective To investigate the relationship between the expression of GATA6 mRNA and the occu-rrence of congenital heart disease(CHD). Methods A total of 60 cases of CHD (CHD group)were diagnosed at Obstetrical Department,Rizhao City Maternal and Child Health Hospital,and 60 cases of normal pregnancy women (control group)were collected in March 2013 to May 2017. The expression levels of mRNA of GATA6 in the peripheral blood of pregnant women in 2 groups were detected by using real-time quantitative PCR (qRT-PCR)technique,and the relevant information of 2 groups of mothers before and during pregnancy was collected so as to investigate the rela-tionship between mRNA and GATA6 in CHD. Results There were no significant differences in age,body mass index (BMI),passive smoking,alcohol consumption,pregnancy medication,pregnancy-induced hypertension,high -density lipoproteincholesterol (HDL-C),total cholesterol (TC),triacylglycerol (TG)between pregnant women in the congenital heart disease group and the control group (all P > 0. 05). The CHD group showed a statistical difference significance in the rate of taking folic acid(81. 67％)and gestational diabetes incidence(8. 33％),serum level of LDL-C[(3. 59 ± 0. 46)mmol/ L]compared with the healthy control group [96. 67％,0,(3. 20 ± 0. 44)mmol/ L] (all P < 0. 05). The expression level of GATA6 mRNA in peripheral blood of patients with congenital heart disease (0. 014 ± 0. 005)was significantly lower than that of the control group (0. 129 ± 0. 031),and the difference was statis-tically significant (t = 28. 386,P = 0. 000). Logistic regression analysis showed that the level of in GATA6 mRNA ex-pression increased in the peripheral blood and folic acid intake were protective factors for CHD (all P < 0. 05)during pregnancy;while fetal gestational diabetes,elevated levels of LDL-C are risk factors for congenital heart disease (all P < 0. 05)fetus. Conclusion Down regulation of GATA6 mRNA expression in the maternal peripheral blood is one of the independent risk factors for fetal congenital heart disease.

Comparison of real-time quantitative PCR and 16S rDNA sequencing for detection of female vaginal microbiome / 上海交通大学学报（医学版）

Objective·To compare the differences in the composition of female vaginal flora by real-time quantitative PCR and 16S rDNA sequencing.Methods·Forty-nine healthy reproductive women less than 45 years old were selected.Specimens were collected from posterior fornix.DNA were extracted and the microbiome were ananlyzed by both 16S rDNA V1V2 region sequencing and qPCR of 22 selected genes.The results detected by two methods were compared.Results·According to the classification standard of vaginal community state type (CSTs),qPCR analysis showed that 35 out of 49 samples were dominated by Lactobacillus species,among them,type Ⅰ (Lactobacillus crispatus,9),type Ⅲ (Lactobacillus iners,24),type Ⅳ (no Lactobacillus as the dominant bacteria,12),type Ⅴ (Lactobacillusjensenii,2).16S rDNA V1V2 region sequence analysis showed that of the 49 samples,13 belonged to type Ⅰ,type Ⅱ (1),type Ⅲ (23),type Ⅳ (8),type Ⅴ (2).Two methods of vaginal flora classification were consistent for 38 cases,consistent rate was 77.6％.Conclusion·Two methods analysis of vaginal flora showed the different results.If qPCR was used to classify the vaginal microbiome,it was necessary to consider the influence of relevant technical factors on the results.