Detection of circulating hepatocellular carcinoma cells in peripheral venous blood by reverse transcription-polymerase chain reaction

Objective: To detect circulating hepatocellular carcino-ma by demonstrating hepatocellular carcinoma cells orhepatocyte-associated mRNA in the nuclear cell com-ponent of peripheral blood (PBL).Methods: Peripheral blood (5 ml) samples were ob-tained from 93 patients with hepatocellular carcinoma(HCC) and from 33 control subjects (9 with liver cir-rhosis after hepatitis B,14 with chronic hepatitis B,10with normal liver function). To identify HCC cells inperipheral blood, liver-specific human alpha-fetopro-tein (AFP) mRNA was amplified from total RNA ex-tracted from whole blood by reverse transcription-polymerase chain reaction.Results: AFPmRNA was detected in 50 blood samplesfrom the HCC patients (50/93, 53.8%). In contrast,there were no clinical control patients whose samplesshowed detectable AFPmRNA in PBL. The presence ofAFPmRNA in blood seemed to be correlated with thestage (by TNM classification) of HCC, the serum AFPvalue, and the presence of intrahepatic metastasis,portal vein thrombosis, tumor diameter and/or distantmetastasis. In addition, AFPmRNA was detected in theblood of 21 patients with metastasis at extrahepaticorgans (100%) in contrast to 29 (40.3%)of 72 pa-tients without metastasis.Conclusion: The presence of AFPmRNA in peripheralblood may be an indicator of malignant hepatocytes,which might predict hematogenous spreading metasta-sis of tumor cells in patients with HCC.

Quantification of Porcine Follicle-stimulating Hormone Receptor Messenger Ribonucleic Acid by Reverse Transcription competitive Polymerase Chain Reaction

An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.

Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription

Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

Detection of hepatocellular carcinoma cells in the peripheral blood with reverse--transcription polymerase chain reaction

In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samples of 113 cases of HCC and 69 controls (including 30 cases of liver cirrhosis, 9 cases of metastatic liver cancer and 30 normal subjects). 20/43 (46. 5% ) cases of HCC and 2/30 (6. 7% ) cases of liver cirrhosis are positive and the cases of nletastatic liver cancer and normal controls were negative for human AFP(hAFP) rnRNA. The presence of hAFP mRNA in the peripheral blood seems to be correlated with intrahepatic and distant nletastasls of HCC and portal vein thrombosis. It is concluded that the presence of hAFP mRNA in the peripheral hloocl is an indicator of circulating HCC cells and can be used to diagnose the rnetastasisof HCC through henlatogenous route and RT-PCR amplification of hAFP mRNA is a sensitive and specificprocedure for detecting circulating cells of HCC.

Small Amplicons Mutation Library for Vaccine Screening by Error-Prone Polymerase Chain Reaction

Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.

Quantification of mRNA Levels by Fluorescently Labelled Reverse Transcription Competitive PCR

A reproducible,quantitative,non-radioactive method for the analysis of mRNA expression is described.After RNA preparation and cDNA synthesis,the cDNA was co-amplified with an internal standard in the same PCR system.The PCR products containing both targen and internal standard amplificates were electrophoresed and detected on an ABI 377 DNA Sequencer.For each sample,β-actin was also quantified by an identical procedure to compensate for relative differences between samples in the integrity of the individual RNA samples and for variations in reverse transcription.Due to the linear relationship between cDNA content and PCR product ratio of target cDNA template and competitive standard,a single PCR reaction was sufficient for quantification of a sample.The experimental results showed that the method is a mRNA quantitative RT-PCR method with high sensitivity and good reproducibility.It can be used in large-scale accurate quantitative analyses of mRNA expression of any gene.

Detection of SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction

OBJECTIVE: To assess the feasibility of detecting SYT-SSX fusion transcripts in paraffin-embedded tissues of synovial sarcoma by reverse transcription-polymerase chain reaction (RT-PCR). METHODS: RT-PCR was used to amplify the SYT-SSX fusion transcripts using archival formalin-fixed paraffin-embedded tumor specimens from a series of 37 synovial sarcoma cases. To investigate the specificity of the SYT-SSX fusion transcripts, a variety of non-synovial sarcoma tumors were included in the study as negative controls. The detected messages derived from fusion genes were confirmed by subsequent sequence analysis. RESULTS: SYT-SSX fusion transcripts were detected in 33 of 37 (89.2%) synovial sarcomas. None of the 34 cases of non-synovial sarcoma tumors showed amplified products of SYT-SSX fusion transcripts, although PBGD mRNA was detected in all specimens. Among 33 SYT-SSX-positive synovial sarcomas, 22 tumors had an SYT-SSX 1 fusion transcript, whereas 6 tumors had an SYT-SSX2 fusion transcript. Fusion types can not be distinguished in the remaining 5 cases. There was a significant relationship between SYT-SSX fusion type and histologic subtype. All 10 biphasic synovial sarcomas had the SYT-SSX1 fusion, whereas all tumors with SYT-SSX2 were of monophasic morphology (P

SEQUENCE ANALYSIS OF THE NS5 REGION OF GBVC/HGV AND DETECTION OF THE VIRUS BY REVERSE TRANSCRIPTASE PCR

GBV C/HGV is a newly identified virus associated with human hepatitis In this study, the nucleotide sequences of the partial NS5 gene of GBV C/HGV derived from sera of 8 Chinese patients were determined The nucleotide homology among the 8 isolates were 92% on average On the basis of sequence analysis, two sets of oligonucleotide primers derived from highly conserved region of GBV C/HGV NS5 gene were designed to establish both sensitive and specific nested PCR for detection of GBV C/HGV RNA 253 Chinese patients were examined for the virus RNA GBV C/HGV RNA positive rates in patients infected with HBV, HCV and patients with chronic non B,non C hepatitis were 18 4%, 19 8% and 8 9% respectively This result suggested that HBV,HCV and GBV C/HGV shared the same transmission risk factors 8 patients with GBV C/HGV and HCV coinfection were retrospectively observed for the response to interferon Coinfection with GBV/HGV did not negatively influence the responsiveness of HCV, and GBV C/HGV was sensitive to interferon to a certain degree

Comparison of the Tellgenplex HPV DNA test with the PCR-reverse dot blot assay for human papillomavirus genotyping

Objective: To access the performance of the Tellgenplex human papillomavirus(HPV) DNA test compared to the polymerase chain reaction-reverse dot blot(PCR-RDB) assay for the HPV genotyping.Methods: Sixty cervical swab samples were genotyped by the Tellgenplex HPV DNA test and the PCR-RDB assay.The Tellgenplex HPV DNA test and the PCR-RDB assay can detect 26 and 23 HPV genotypes, respectively.Each sample showed discrepancy was genotyped using sequencing.Results: The percent agreement between the two tests ranged from 83.3% to 100.0% according to different genotype.This showed perfect agreement(>0.81) for high-risk HPV genotypes(35, 39, 45, 53, 56, 59, 66, 68, and 82), substantial agreement(>0.65) for high-risk HPV genotypes(16, 18, 33, 52, and 58) and low-risk HPV genotype 43 between the two assays by the kappa analysis.The positive rates of the two assays for frequent HPV genotypes(16, 35, 39, 45, 52, 53, 58, 59, 66, and 82) were not statistically different, but the PCR-RDB assay showed higher positive rates than the Tellgenplex HPV DNA test for HPV genotypes 81(P<0.05).As for more than 10 positive results by the Tellgenplex HPV DNA test and/or the PCR-RDB assay, the PCR-RDB assay showed higher relative sensitivity and specificity than the Tellgenplex HPV DNA test for the three HPV genotypes(16, 52, and 81).All HPV genotypes that can be detected by only the Tellgenplex HPV DNA test(HPV genotypes 44 and 55) were confirmed by sequencing.Conclusions: In conclusion, our results demonstrated that the PCR-RDB assay which can detect more multiple HPV genotypes in each specimen shows higher relative sensitivity and specificity than the Tellgenplex HPV DNA test, which makes it a better option for routine clinical use.

A rapid reverse dot blot assay for all 18 β-thalassemia mutations in Chinese population

A set of allele-specific oligonucleotide (ASO) probes used for detecting all 18 β-tha-lassemia mutations found in Chinese was immobilized on two strips of Biodyne C membrane;one containing 7 pairs of oligonucleotide probes specific for the most commonly found mutant al-leles,and the other containing the remaining 11 pairs of ASO_s specific for the less commonlyfound.The membranes were hybridized with β-globin sequences amplified by polymerase chainreaction (PCR) with biotinylated primers,and then treated with Streptavidin-POD conjugateand substrates for color development.The method has been applied successfully to the detectionof all 18 Chinese β-thalassemia mutations and prenatal diagnosis of two high-risk pregnancies ofβ-thalassemia.Patients with homozygous,heterozygous and compound heterozygous alleles ofthese mutations and normal individuals could be easily distinguished by the present method.Us-ing the immobilized-probe format (reverse dot blot),it was able to screen simultaneously multi-ple β-thalassemia mutations of a DNA sample by performing hybridization only once.This assayis simple,rapid and independent of radio-isotopes and can be appplied for all 18 β-thalassemiamutations so far found in Chinese population.It is considered that this method may be usefulfor gene frequency investigation of large numbers of β-thalassemia DNA samples and used as aroutine method in the clinic laboratory.

Investigation of SRY Gene in Sex Reversed Syndrome Patients

SRY (sex-determining region Y chromosome) is considered as a strong candidste for the TDF (testis determining factor) and has been cloned following another candidate ZFY (zinc finger protein gene). In this study,eight cases of sex revelsal, including four 46, XX males and four 46, XY females aere examined for the presence of SRY sequence and a Y-repeated DNA locus. Our data indicated that the genownic DNA of the four classical 46,XX males had the SRY sequences. On the other hand, both SRY sequences and Ye repeated DNA sequences were present in all four 46, XY females.These resultssuavest that SAY sequences were responsible for the sexraversal of 46, XX males Whereas there may be other genetic mechanisms forthe sex reversal of 46, XY females Without the lack of SRY sequences.

复合探针实时荧光RT-PCR法检测小儿上呼吸道感染甲型流感病毒的价值

目的 分析小儿上呼吸道感染甲型流感病毒应用复合探针实时荧光反转录聚合酶链反应(RT-PCR)法检测的临床价值。方法选择2023年1月至2023年12月流感监测信息系统两家监测点上呼吸道感染甲型流感病毒感染的患儿80例监测标本进行回顾性分析,均开展复合探针实时荧光RT-PCR法检测,分析其诊断价值。结果 根据监测标本最终诊断结果显示,阳性标本68例、阴性标本12例。经复合探针实时荧光RT-PCR法检出67例,检出率为83.75%,敏感度为95.59%、特异度为83.33%、准确度为93.75%、阳性结果预测值为97.01%、阴性结果预测值为76.92%;批间批内变异系数均小于5%。结论 小儿上呼吸道感染甲型流感病毒应用复合探针实时荧光RT-PCR技术具有较高的敏感度、特异度及准确度,且检查结果快速,可为小儿上呼吸道感染甲型流感病变提供可靠的诊断,有利于制订合理的治疗方案。

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。

帕利亚姆病毒实时荧光定量RT-PCR检测方法的建立与应用

本研究拟建立帕利亚姆病毒(Palyam virus,PALV)血清型特异性实时荧光定量RT-PCR(qRT-PCR)方法用于临床样本或媒介中PALV血清型鉴定。根据我国流行PALV毒株的基因节段2序列,设计扩增引物和TaqMan探针,建立PALV血清型特异型qRT-PCR方法,对方法的特异性、灵敏性与重复性进行评估;以我国分离的28株PALV和90份核酸阳性血液样本评估检测方法的可靠性;利用建立的方法对采集库蠓样本中携带的PALV进行血清型鉴定。结果显示,建立的PALV血清型qRT-PCR检测方法具有良好的特异性与灵敏性,可检出核酸拷贝数下限在22至28 copies·μL^(-1)。对28株PALV的qRT-PCR检测结果与病毒测序鉴定结果一致;对PALV不同感染阶段哨兵动物血液(90份)中的qRT-PCR鉴定结果与分离病毒的血清型鉴定结果一致;建立的方法可准确鉴定库蠓中携带PALV的血清型。本研究建立的PALV血清型qRT-PCR定型方法具有良好的特异强、敏感性与重复性,可用于PALV感染动物与媒介中PALV血清型的鉴定,具有良好的应用价值。

新型冠状病毒亚基因组RNA检测方法的建立及性能评估

目的建立检测新型冠状病毒(新冠病毒)亚基因组RNA(subgenomic RNA,sgRNA)的方法,并对所建立的方法进行性能评估。方法根据新型冠状病毒sgRNA序列设计引物和探针,建立检测sgRNA的实时荧光逆转录PCR(reverse transcription polymerase chain reaction,RT-PCR)方法,对所建立的方法进行优化,包括引物、探针的浓度及比例、延伸温度、反应体积和模板量。并对所建立的方法进行性能评估,包括最低检测限、灵敏度、特异性和重复性。对临床样本进行新冠病毒sgRNA和基因组RNA(genomic RNA,gRNA)检测,并对检测结果进行分析。结果建立了检测新冠病毒sgRNA的RT-PCR方法。所建方法的最低检测限为100 copies/mL,对常见病原体的检测结果均为阴性。对高、中、低浓度样本sgRNA进行检测,CV均<5%。115例疑似新冠病毒感染者的咽拭子样本sgRNA为阳性(115/330,阳性率为34.85%)。gRNA-N Ct值<30时,sgRNA-N的阳性率为100.00%;gRNA-N的Ct值介于30~32时,sgRNA-N的阳性率为68.75%;gRNA-N的Ct值介于32~35时,sgRNA-N的阳性率为44.44%;gRNA-N的Ct值>35时,sgRNA-N均为阴性。结论建立了新冠病毒sgRNA的RT-PCR检测方法,方法灵敏、特异、重复性好。

新型冠状病毒Omicron I1566V突变位点MS2噬菌体病毒样颗粒的构建

目的构建新型冠状病毒Omicron I1566V突变位点MS2噬菌体病毒样颗粒。方法选取并合成带有6×His标签的MS2噬菌体的衣壳蛋白(CP)、成熟蛋白(A蛋白)及Omicron I1566V突变基因序列,插入pACYCDuet-1质粒构建重组载体,通过原核系统诱导表达目的蛋白,纯化重组蛋白后利用透射电镜对蛋白质进行物理表征,最后通过反转录聚合酶链反应(RT-PCR)检测病毒样颗粒的热稳定性及耐核酸酶水解能力。结果成功构建包含有6×His标签的CP、A蛋白和Omicron I1566V突变基因序列的重组载体,经限制性内切酶BamHⅠ和KpnⅠ酶切鉴定和测序验证,结果均与预期相符。经诱导并纯化后,通过电镜观察到了大小均匀、直径为23~28 nm的病毒样颗粒,该病毒样颗粒经核酸酶消化后可在37℃条件下稳定储存20 d以上。结论该研究成功利用MS2噬菌体的CP和A蛋白构建了Omicron I1566V突变位点病毒样颗粒,为该突变位点的RT-PCR检测体系提供了可靠的质量保障。

基于ITS基因序列的羊源食道口线虫PCR检测方法建立及应用

为了能对羊源食道口线虫病进行特异性诊断,基于食道口线虫ITS基因序列设计特异性引物,建立食道口线虫的特异性PCR检测方法,对其特异性、敏感性以及在临床羊粪便样本中食道口线虫虫卵的检测效果进行验证。结果显示,在对食道口线虫、仰口线虫、毛尾线虫、捻转血矛线虫等9种线虫的阳性DNA模板进行扩增时,只有在食道口线虫DNA阳性模板中扩增出650 bp的目的条带,而在其他线虫的DNA模板中无条带出现;将食道口线虫原始质粒模板进行10倍梯度稀释后进行敏感性检测,该PCR最低检出浓度为90 copies/μL;利用所建立的PCR检测方法对临床200份羊粪便样本进行检测,结果检出食道口线虫的阳性率为32%,高于显微镜镜检的阳性检测结果(阳性率为31%),但二者无显著性差异。研究表明,该研究所建立的食道口线虫PCR检测方法能够特异性地检测食道口线虫及其虫卵的阳性DNA样本,且敏感性良好,该方法为食道口线虫的种类鉴别以及食道口线虫病的诊断提供了一种可供选择的技术。

菊芋HtMYB2基因VIGS体系构建与功能验证

颜色作为一种重要的农艺性状,影响作物的价值。紫皮菊芋与白皮菊芋相比,具有较好的外观颜色、营养价值和商品价值。本研究旨在探究菊芋块茎表皮颜色形成的潜在机制。通过前期转录组测序技术对紫皮和白皮菊芋块茎表皮分析,筛选得到与花青素合成代谢相关转录因子HtMYB2。本研究以紫皮菊芋品种为材料,利用VIGS技术将HtMYB2进行基因沉默,对其功能进行验证。结果表明侵染后的菊芋块茎表皮颜色变浅,花青素含量显著下降为15.34 mg/g;同时RT-PCR结果显示HtMYB2基因表达量为2.06,与对照组相比显著降低,沉默效率为73.96%,推测HtMYB2基因的沉默使菊芋块茎中花青素基因的表达受阻,花青素合成功能减弱,导致花青素含量降低,表明HtMYB2基因在花青素合成途径中起正调控作用。本研究为菊芋块茎表皮花青素的生物合成机理提供理论依据,并进一步揭示了该基因在调控块茎颜色和花青素合成途径中的重要性。

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.