Confusing finding of quantitative fluorescent polymerase chain reaction analysis in invasive prenatal genetic diagnosis:A case report

BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.

Primary application of a real-time quantitative polymerase chain reaction for the detection of human breast cancer related novel gene-Metadherin expression

Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.

Real-time Fluorescence PCR Method for Detection of Burkholderia glumae from Rice

Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It＇s quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×10^4 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1：100.

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus(ZIKV)and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction(ddPCR) and real-time quantitative PCR(RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold(Ct) value was linear from 10~1 to 10~8 copy/l L, with a standard curve R^2 of 0.999 and amplification efficiency of 92.203%;however, a concentration as low as 1 copy/l L could not be detected. In comparison with RT-qPCR, the dd PCR method resulted in a linear range of 10~1–10~4 copy/l L and was able to detect concentrations as low as 1 copy/l L. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples(above 10~1 copy/l L),while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.

Comparison of Abbott and Da-an real-time PCR for quantitating serum HBV DNA

AIM: To compare the performance of the Da-an real-time hepatitis B virus (HBV) DNA assay and Abbott RealTime HBV assay.

Rapid prenatal diagnosis of trisomy 21 by fluorescent quantitative multiplex polymerase chain reaction

Trisomy 21, also named Down syndrome was the most frequent autosomal aneuploidy and the most common cause of mental retardation. Fifty percent patients had congenital heart malformation. Every 20 minutes one case of trisomy 21 was born, and the incidence rate was 1 in 600 to 800 newborns in China.1 In two thirds of cases with trisomy 21, there was a spontaneous abortion, so the actual incidence was higher than that obtained postnatally.

Reference genes for quantitative RT-PCR data in gastric tissues and cell lines

AIM:To evaluate the suitability of reference genes in gastric tissue samples and cell lines.METHODS:The suitability of genes ACTB,B2M,GAPDH,RPL29,and 18S rRNA was assessed in21 matched pairs of neoplastic and adjacent nonneoplastic gastric tissues from patients with gastric adenocarcinoma,27 normal gastric tissues from patients without cancer,and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR).The ranking of the best single and combination of reference genes was determined by NormFinder,geNorm,BestKeeper,and DataAssist.In addition,GenEx software was used to determine the optimal number of reference genes.To validate the results,the mRNA expression of a target gene,DNMT1,was quantified using the different reference gene combinations suggested by the various software packages for normalization.RESULTS:ACTB was the best reference gene for all gastric tissues,cell lines and all gastric tissues plus cell lines.GAPDH+B2M or ACTB+B2M was the best combination of reference genes for all the gastric tissues.On the other hand,ACTB+B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines.According to the GenEx software,2 or 3 genes were the optimal number of references genes for all the gastric tissues.The relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes.The level of expression of DNMT1 in neoplastic,adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH+B2M(P=0.32),ACTB+B2M(P=0.61),or GAPDH+B2M+ACTB(P=0.44).CONCLUSION:GAPDH+B2M or ACTB+B2M is the best combination of reference gene for all the gastric tissues,and ACTB+B2M is the best combination for the cell lines tested.

基于直扩RT-PCR技术的寨卡病毒快速检测方法的建立

目的建立基于直扩实时荧光定量逆转录聚合酶链反应(RT-PCR)技术的寨卡病毒快速检测方法。方法采用特殊功能的DNA聚合酶,以及优选PCR增强剂,以此建立直扩RT-PCR技术的寨卡病毒快速检测5种样本(全血、血清、唾液、咽拭子和尿)的方法。结果5种样本检测限分别为血清103 PFU/mL,尿、咽拭子和唾液102 PFU/mL,全血104 PFU/mL,标准曲线的拟合优度的可决系数均在0.98以上,扩增效率均在90%~110%;寨卡病毒核酸成功扩增,非寨卡病毒核酸均未能扩增;尿、全血和唾液样本的重复性实验中106 PFU/mL和102 PFU/mL两个浓度的6个重复Ct值的变异系数均<5%。该研究建立的直扩RT-PCR技术的寨卡病毒检测方法与常规RT-PCR技术的检测结果一致,8个寨卡病毒样本,均只检测出2个血清样本,其余62个非寨卡病毒样本及12个阴性样本均未得到扩增。结论成功建立基于直扩RT-PCR技术的寨卡病毒快速检测方法,该方法简便快捷且灵敏度高、特异度强。

Influences of bracket bonding on mutans streptococcus in plaque detected by real time fluorescence-quantitative polymerase chain reaction

Background Enamel demineralization occurs frequently during orthodontic treatment. In this study, we evaluated the changes of the density of mutans streptococcus （MS） in plaque after bracket bonding and using fluoride adhesive on maxillary incisors by real time fluorescence-quantitative polymerase chain reaction （RT-FQ PCR）.Methods The study was designed as a self-paired test. Brackets were bonded with fluoride adhesive on the left side, while non-fluoride adhesive on the right side for each patient. Plaque samples were taken from the surfaces around the brackets of four maxillary incisors before brackets bonding and after the bonding 4 weeks later. The amount of MS was measured by RT-FQ PCR. The data obtained were analyzed statistically using the SPSS 11.5 version and the alpha level was set at 0. 05 （ 2-tailed）.Results The amount of MS in plaque increased significantly after bracket bonding （ P 〈 0.01 ）, whereas no significant differences were observed among four maxillary incisors both before and after brackets bonding （P 〉 0. 05 ）, and among the incisors using and not using fluoride adhesive （ P 〉 0. 05 ）.Conclusions The increase of the density of MS in plaque after bracket bonding is one of the etiological factors for enamel demineralization in orthodontic patients. The result of this study did not support what we observed clinically that the incidence of enamel demineralization for lateral incisors was higher than that for central incisors. Using fluoride adhesive for bonding did not affect the amount of MS in plaque in our study. Further study is needed.

Establishment of a new quantitative detection approach to adefovir-resistant HBV and its clinical application

AIM:To establish the more feasible and sensitive assessment approach to the detection of adefovir (ADV) resistance-associated hepatitis B virus (HBV) quasispecies.METHODS: Based on the characteristics of rtA181V/T and rtN236T mutations, a new approach based on real-time fluorescent quantitative polymerase chain reaction (RT-PCR) was established for the detection of ADV-resistant HBV quasispecies, total HBV DNA, rtA181 and rtN236 mutations in blood samples from 32 chronic hepatitis B (CHB) patients with unsatisfactory curative effect on ADV and compared with routine HBV DNA sequencing.RESULTS: Both the sensitivity and specificity of this new detection approach to ADV-resistant HBV quasispecies were 100%, which were much higher than those of direct HBV DNA sequencing. The approach was able to detect 0.1% of mutated strains in a total plasmid population. Among the 32 clinical patients, single rtA181 and rtN236T mutation and double rtA181T and rtN236T mutations were detected in 20 and 8, respectively, while ADV-resistant mutations in 6 (including, rtA181V/T mutation alone in 5 patients) and no associated mutations in 26.CONCLUSION: This new approach is more feasible and efficient to detect ADV-resistant mutants of HBV and ADV-resistant mutations before and during ADV treatment with a specificity of 100% and a sensitivity of 100%.

Quantitative Analysis of ATP Sulfurylase and Selenocysteine Methyltransferase Gene Expression in Different Organs of Tea Plant (<i>Camellia sinensis</i>)

Tea plant (Camellia sinensis) has unique biological features for the study of cellular and molecular mechanisms, an evergreen broad-leaved woody plant which can accumulate selenium in soil abundant of Selenium. Expression of the genes related to Selenium (Se) metabolism is an adaptation to the soil environment for a long period. The purpose of the present study was to explore if there exist differences of expression about these genes in tea plant between growing in Selenium-abundant and normal soil. A quantitative real-time reverse transcription polymerase chain reaction (Q-RT-PCR) assay was done for quantification of ATP sulfurylase (APS) and selenocysteine methyltransferase (SMT) mRNA normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in tea plant. Young leaves, mature leaves and tender roots from tea plants growing in soil abundant of Selenium were respectively obtained from Shitai County, Anhui Province, and also the relevant materials of the selenium un-enriched tea plant planted at agricultural garden of Ahui Agriculture University were taken as control for real-time PCR analysis. The results showed that APS1, APS2 and SMT expression levels for either young or mature leaves in selenium-enriched tea plant were lower than that in ordinary (selenium un-enriched) tea plant. In contrast, the APS1, APS2 and SMT expression level of roots in selenium-enriched tea plant were all higher than that in ordinary tea plant. APS1 gene expression level of roots in selenium-enriched tea plant was about 1.6 times higher than that in the ordinary tea plant, APS2 gene expression level was about 4.8-fold higher than that in the ordinary tea plant, SMT gene expression level was about 3.3 times higher than that in the ordinary tea plant. Among various tissues of selenium-enriched tea plant, APS1 gene relative expression level of young leaves was similar to or slightly higher than mature leaves, and the one of roots was the lowest among them;APS2 gene relative expression level of young leaves was similar to or slightly higher than the roots, and the one of mature leaves was the lowest among them;SMT gene relative expression level of young leaves was similar to or slightly higher than mature leaves, and the one of roots was the highest among them. Our results suggest that there existed correlation between selenium and expression levels of these genes.

Quantitative analysis of a panel of gene expression in prostate cancer——with emphasis on NPY expression analysis

Objective： To investigate molecular alterations associating with prostate carcinoma progression and potentially provide information toward more accurate prognosis/diagnosis. Methods： A set of laser captured microdissected （LCM） specimens from 300 prostate cancer （PCa） patients undergoing radical prostatectomy （RP） were defined. Ten patients representing ＂aggressive＂ PCa, and 10 representing ＂non-aggressive＂ PCa were selected based on prostate-specific antigen （PSA） recurrence, Gleason score, pathological stage and tumor cell differentiation, with matched patient age and race between the two groups. Normal and neoplastic prostate epithelial cells were collected with LCM from frozen tissue slides obtained from the RP specimens. The expressions of a panel of genes, including NPY, PTEN, AR, AMACR, DD3, and GSTP1, were measured by quantitative real-time RT-PCR （TaqMan）, and correlation was analyzed with clinicopathological features. Results： The expressions of AMACR and DD3 were consistently up-regulated in cancer cells compared to benign prostate epithelial cells in all PCa patients, whereas GSTP1 expression was down regulated in each patient. NPY, PTEN and AR exhibited a striking difference in their expression patterns between aggressive and non-aggressive PCas （P=0.0203, 0.0284, and 0.0378, respectively, Wilcoxon rank sum test）. The lower expression of NPY showed association with ＂aggressive＂ PCas based on a larger PCa patient cohort analysis （P=0.0037, univariate generalized linear model （GLM） analysis）. Conclusion： Despite widely noted heterogeneous nature of PCa, gene expression alterations ofAM,4CR, DD3, and GSTP1 in LCM-derived PCa epithelial cells suggest for common underlying mechanisms in the initiation of PCa. Lower NPY expression level is significantly associated with more aggressive clinical behavior of PCa; PTEN and AR may have potential in defining PCa with aggressive clinical behavior. Studies along these lines have potential to define PCa-associated gene expression alterations and likely co-regulation of genes/pathways critical in the biology of PCa onset/progression.

Prenatal diagnosis of Down syndrome using cell-free fetal DNA in amniotic fluid by quantitative fluorescent polymersase chain reaction

Backgroud Amniotic fluid （AF） supernatant contains cell-free fetal DNA （cffDNA） fragments.This study attempted to take advantage of cffDNA as a new material for prenatal diagnosis,which could be combined with simple quantitative fluorescent polymerase chain reaction （QF-PCR） to provide an ancillary method for the prenatal diagnosis of trisomy 21 syndrome.Methods AF supernatant samples were obtained from 27 women carrying euploid fetuses and 28 women carrying aneuploid fetuses with known cytogenetic karyotypes.Peripheral blood samples of the parents were collected at the same time.Short tandem repeat （STR） fragments on chromosome 21 were amplified by QF-PCR.Fetal condition and the parental source of the extra chromosome could be determined by the STR peaks.Results The sensitivity of the assay for the aneuploid was 93％ （26/28; confidence interval,CI：77％-98％） and the specificity was 100％ （26/26; CI：88％-100％）.The determination rate of the origin of the extra chromosome was 69％.The sensitivity and the specificity of the assay in the euploid were 100％ （27/27）.Conclusions Trisomy 21 can be prenatally diagnosed by the QF-PCR method in AF supernatant.This karyotype analysis method greatly reduces the requirement for the specimen size.It will be a benefit for early amniocentesis and could avoid pregnancy complications.The method may become an ancillary method for prenatal diagnosis of trisomy 21.

Sequence Analysis and Quantitative Detection of Norwalk-like Viruses in Cultured Oysters of China

We isolated 4 Norwalk-like viruses （NLVs） contaminated oysters from 33 Chinese oysters collected from local commercial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase （RdRp） region of NLVs genomes with RT-PCR, the open reading frame 1 （ORF 1） of the RdRp was sequenced and subjected to multiple-sequence alignment. The results showed that NLVs in the four isolates belong to genogroup Ⅱ. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts （copies） of NLVs in positive patient＇s fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9× 10^8, 1.25× 10^8 and 4.7× 10^1 respectively. The detecting limit of NLVs was 1 × 10^1 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future.

饮食习惯与肥胖患儿性早熟的相关性分析

目的探讨饮食习惯与肥胖患儿性早熟的相关性分析。方法选取苏州市吴中人民医院2019年3月至2022年3月期间收治的72例性早熟肥胖患儿作为观察组,另选取同期体检的健康肥胖儿童71例作为常规组。采用实时-逆转录荧光定量聚合酶链反应(RT-qPCR)检测两组外周血miR-125b水平,分析患儿饮食习惯。通过比较两组肥胖儿童的外周血miR-125b、饮食习惯,采用Logistic回归分析法分析外周血miR-125b、饮食习惯与肥胖患儿性早熟的关系。结果观察组外周血miR-125b表达水平高于常规组,差异有统计学意义(P<0.05)。观察组饮食没规律、荤多素少、高添加剂食品占比均高于常规组,差异有统计学意义(P<0.05)。观察组女性患儿、不良饮食习惯占比高于常规组,且经多因素分析显示外周血miR-125b表达水平、女性、不良饮食习惯是肥胖患儿性早熟的独立危险因素,差异有统计学意义(P<0.05)。结论肥胖患儿性早熟外周血miR-125b表达水平高于健康肥胖儿童,不良饮食习惯高于健康肥胖儿童,外周血miR-125b表达水平偏高、不良饮食习惯偏低均为肥胖患儿性早熟的影响因素。

鼠李糖乳杆菌HN001菌株水平快速定量方法的建立及应用

目的应用实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,qPCR),建立鼠李糖乳杆菌HN001菌株水平的快速定量检测方法。方法通过比对鼠李糖乳杆菌HN001的近缘菌株的基因组,筛选出鼠李糖乳杆菌HN001菌株水平的特异基因,以菌株水平的特异基因为靶标设计引物及探针,优化反应体系和条件,建立鼠李糖乳杆菌HN001株水平的qPCR检测方法。对方法的特异性、灵敏度、检出限进行验证,并采用已建立的qPCR方法进行模拟添加样品和实际样品的检测。结果所建立的方法特异性强,与近缘菌株无交叉反应,鼠李糖乳杆菌HN001纯培养液检出限为10^(2)CFU/mL,灵敏度可达到650 copies/μL,可在2 h内完成样品检测。对模拟添加样品以及实际样品检测中qPCR和平板计数法结果的对数值进行显著性分析检验,两种方法的测定值结果均无显著性差异(P>0.05)。结论本研究建立的qPCR检测方法可有效应用于益生菌产品中鼠李糖乳杆菌HN001菌株水平定量检测,具有快速、灵敏、准确的特点。

实时荧光定量RT-PCR分析非小细胞肺癌SATB1的表达和临床病理意义

目的检测非小细胞肺癌组织中SATB1 mRNA的表达,探讨SATB1表达与非小细胞肺癌发生、发展的关系及其临床病理意义。方法用TRIZOL提取非小细胞肺癌组织和正常肺组织总RNA后,将其反转录为cDNA,以实时荧光定量RT-PCR方法检测非小细胞肺癌组织及正常肺组织中SATB1 mRNA的表达,分析SATB1基因表达与临床病理参数的相关性。结果癌组织中SATB1 mRNA表达量为正常组织13倍,两者差异显著(P<0.001),其中有、无转移组分别为正常组织23.63倍和5.57倍。结论SATB1 mRNA的表达水平可能与非小细胞肺癌发生发展及淋巴转移有关,有望成为判断非小细胞肺癌预后的一个指标。

基于微流控芯片技术的创伤弧菌特异性引物的筛选及验证

目的探讨适用于创伤弧菌的特异性引物,通过微流控芯片技术进行一站式检测,为快速检测出病原体奠定基础。方法通过NCBI网站获取靶基因序列,采用MEGA7.0软件对齐后设计出19对引物,BLAST确定引物的特异度,再通过引物性能、灵敏度、快速变温实验筛选适用于微流控的引物,最后对引物的特异度与灵敏度进行评价。结果成功筛选出1对适用于微流控的引物vvhA10,微流控检测结果发现其特异度与灵敏度较高。结论筛选出适用于微流控芯片的引物,用于全自动微流控检测可以满足应急检测或现场快速检测等方面的使用需求。

应用实时荧光定量RT-PCR法建立肝癌分子诊断指数

目的:筛选肝细胞癌(hepatocellular carcinoma,HCC)中表达水平较正常肝细胞差异大、特异性好的基因,建立肝癌分子诊断指数,以期从分子病因学角度实现对肝癌的早期诊断.方法:随机选择38例手术切除组织标本,其中正常肝5例、肝炎4例、肝硬化(LC)12例、HCC和癌旁组织17例,实时荧光定量RT-PCR检测9个基因的表达,以管家基因G3PDH为对照,2-△△CT法计算目的基因相对表达量,依据表达异常的基因个数建立分子诊断指数.结果:GPC3等6个基因在正常肝、肝炎、LC与HCC组的两组或多组间存在差异(P<0.05);GPC3、E2F1从肝炎组到LC、HCC组呈递增趋势,HGF、CLDN10在LC组表达量升高,而在HCC组明显下降,PTEN、PRDM2、MGMT从肝炎组到LC、HCC组呈递减趋势;9个基因在LC组分子诊断指数平均值为1.58,在HCC组平均值为5.24,二者差异显著;GPC3、E2F1、MMP2从癌旁到癌呈递增趋势,CLDN10、HGF、PTEN、DLC1、PRDM2、MGMT从癌旁到癌呈递减趋势.结论:通过筛选更多基因的组合分析,分子诊断指数有望成为鉴别诊断早期肝癌的一种有效方法.

实时荧光定量RT-PCR检测结直肠癌中外周血端粒酶逆转录酶mRNA的表达及其临床意义

目的:建立外周血端粒酶逆转录酶(hTERT)mRNA表达量检测的实时荧光定量逆转录聚合酶链反应(RT-PCR)系统,检测结直肠癌患者hTERT mRNA的表达并探讨其对结直肠癌微转移的诊断价值.方法:用实时荧光定量RT-PCR技术检测53例结直肠癌患者和21名健康人的外周血标本hTERT mRNA表达情况,分析其表达与肿瘤临床病理特征的关系并应用接受者操作特征曲线(ROC曲线)分析hTERT mRNA检测对结直肠癌的诊断价值.结果:结直肠癌组外周血hTERT mRNA表达水平显著高于正常对照组(t'=7.953,P<0.05),其表达与肿瘤淋巴结转移、血行转移和TNM分期相关(t'/t=2.334,2.149,2.460,均P<0.05).hTERT mRNA诊断结直肠癌的ROC曲线下面积0.91,诊断界值为Ct≤32.结论:应用实时荧光定量RT-PCR技术克服了传统PCR只能定性检测而不能定量检测的缺点;外周血hTERT mRNA可作为诊断结直肠癌微转移的标记物.