TaqMan real-time fluorescent quantitative RT-PCR in detection of macrophage inflammatory protein-2γ mRNA in myocarditis murine

Objective: To study the role of macrophage inflammatory protein (MIP)-2γ in myocarditis pathogenesis in BALB/c mice. Methods: The relationship between the progression of Coxsarckie virus B3(CVB3) viral myocarditis and experimental autoimmune myocarditis and MIP-2γ mRNA expression in mouse was studied by TaqMan real-time fluorescent quantitative RT-PCR. Results: MIP-2γ mRNA expression rose on 3 to 5 d after CVB3 infection, reached peak on 7 d, and returned to normal level until 14 d, which corresponded well with the disease course. The MIP-2γ mRNA expression level rose significantly on the day 18 d after immunization with porcine cardiac myosin, which was consistent with pathological examination. Conclusion: MIP-2γ may be involved in the pathogenesis of myocarditis.

Detection of Survivin mRNA in nasopharyngeal carcinoma by real-time fluorescence quantitative RT-PCR

Objective： To establish the method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in nasopharyngeat carcinoma （NPC） tissues. Methods： The total RNA was extracted from NPC cell line CNE-2 and tissues with Trizol and then been transcribed reversely to cDNA, a method of real time fluorescence quantitative RT-PCR for detecting the expression of Survivin mRNA in NPC tissues had been established, in which chronic nasopharyn-gitis patients＇ nasopharynx tissues treated as control group. Results： The expression of Survivin mRNA all could be detected either in CNE-2 cells, NPC tissues or in chronic nasopharyngitis patients＇ nasopharynx tissues, and there was higher the expression level of Survivin mRNA in NPC tissues than which in chronic nasopharyngitis patients＇ nasopharynx tissues, the difference was significant （P 〈 0.01）. The expression of Survivin mRNA could be detected both in stage Ⅰ ＋ Ⅱ and stage Ⅲ ＋ Ⅳ NPC, and there was no significant difference in relative quantifications of gene expression between these two groups （P 〉 0.05）. There was no relationship between Survivin mRNA expression and age and sex of NPC patients （P 〉 0.05）. Conclusion： Real time fluorescence quantitative RT-PCR is a rapid, effective and high sensitive method for detecting the expression of Survivin mRNA in NPC tissues. The overexpression of Survivin mRNA may play some roles in pathogenesis of NPC.

Detection and clinical significance of multidrug resistance-1 mRNA in bone marrow cells in children with acute lymphoblastic leukemia by real-time fluorescence quantitative RT-PCR

Objective： Multidrug resistance（MDR） is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia（ALL） which may be due to different detection methods. This study was to detect the expression of MDR1 mRNA in bone marrow cells of children with ALL by real-time fluorescence- quantitative reverse transcription polymerase-chain reaction（FQ-RT-PCR）, and combine minimal residual desease（MRD） detection by flow cytometry（FCM） and to study their relationship with treatment response and prognosis of ALL. Methods：The MDR1 mRNA levels in bone marrow cells from 67 children with ALL[28 had newly diagnosed disease, 27 had achieved complete remission（CR）, 12 recurrent] and 22 children without leukemia were detected by FQ-RT-PCR. MRD was detected by FCM. The patients were observed for 9-101 months, with a median of 64 months. Results：Standard curves of human MDR1 and GAPDH genes were constructed successfully. MDR1 mRNA was detected in all children with a positive rate of 100%. The mRNA level of MDR1 was similar among the newly diagnosed ALL group, CR group, and control group（P 〉 0.05）, but significantly higher in the recurrence group than that in newly diagnosed disease group and control group（0.50 ± 0.55 vs. 0.09 ± 0.26 and 0.12 ± 0.23, P〈 0.05）. 54 ALL patients were followed up, and it was found that MDR1 mRNA level was significantly higher in ALL patients within 3 years duration than that of ALL patients with 3-6 years and over 6 years duration（0.63 ± 0.56 vs. 0.11 ± 0.12 and 0.04 ± 0.06, P〈 0.01）. For the 28 children with newly diagnosed disease, the MDR1 mRNA level was similar between WBC 〉 50 ~ 109 group and WBC〈50 × 10^9 group（P〉 0.05）. In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD〉10a group than that in MRD〈10a group（0.39 ± 0.47 vs. 0.03 ± 0.03, P 〈 0.05）. Conclusion：The sensitivity and specificity of FQ-RT-PCR in detecting MDR1 mRNA in bone marrowy cells of children with ALL patients are high. MDR1 mRNA is expressed in children with and without leukemia. MDR1 mRNA is highly expressed in the CR ALL patients with high MRD, recurrence and short duration（within 3 years）. Monitoring MRD and the MDR1 mRNA level might be helpful for individual treatment.

Synchronous Detection of DNA/RNA of Four Shrimp Viruses by Real-time Fluorescence Quantitative RT-PCR

[ Objective] This study aimed to establish a simultaneous detection method of shrimp viruses by real-time fluorescence quantitative RT-PCR, to improve the efficiency of inspection and quarantine. [ Method] A novel real-time fluorescence quantitative RT-PCR assay was established and optimized for simultaneously detecting DNA/RNA of four shrimp viruses （WSSV, IHHNV, TSV and YHV ）. [ Result] The optimized real-time fluorescence quantitative RT-PCR system gener- ated typical amplification curves with high amplification efficiencies （E = 1.06, 1.07, 0.92 and 0.92, respectively）, good hnear relationship （ r = 1 ）, uniform repeatability （ standard deviation = 0.05 - 0.46 ; variation coefficient = 0.26% - 1.62% ） and high sensitivity, exhibiting no significant differences compared with re- al-time fluorescence quantitative PCR （average error of Ct value = 0.04 -0.40; T = 0.53 -2.50; P 〉 0.05 ）. The total detection time was about 1 h. [ Conclusion] The optimized real-time fluorescence quantitative RT-PCR system can be used for rapid detection of WSSV, IHHNV, TSV and YHV.

Establishment and Application of the TaqMan Real-Time Fluorescence Quantitative PCR Detection Assay for Koi Herpes Virus

[ Objective ] This study aimed to establish a rapid and effective quarantine method of Koi herpes virus. [ Method] Primers and corresponding TaqMan probe were designed based on the conserved sequence of Koi herpes virus （KHV） pol-ymerase gene （Sph） to establish a rapid and effective fluorescence quantitative PCR method for Koi herpes virus detection. The cell cultures were detected by using the established fluorescence quantitative PCR assay, and the results were com- pared with that of conventional PCR. [ Result] The sensitivity of fluorescence quantitative PCR was higher than that of conventional PCR. The minimum copy num- ber that could be detected was 1.6 - 102 copies/p.1. The established method was adopted for sample detection, and a reliable diagnostic result could be obtained within 4 h. [Conclusion] The established method is rapid, sensitive, specific and repeatable, which is conducive to the rapid detection of Koi herpes virus. Key words Koi herpes virus; Fluorescence quantitative PCR; Detection

THE DETECTION OF MDR1 GENE EXPRESSION USING FLUOROGENIC PROBE QUANTITATIVE RT-PCR METHOD

Objective: To establish a fluoregenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of the expression of MDR1 gene in tumor cells and to investigate the expression of MDR1 gene in patients with lung cancer. Methods: The fluorogenic quantitative RT-PCR method for detection of the expression of MDR1 gene was established. K562/ADM and K562 cell lines or 45 tumor tissues from patients with lung cancer were examined on PE Applied Biosystems 7700 Sequence Detection machine. Results: the average levels of MDR1 gene expression in K562/ADM cells and K562 cells were (6.86±0.65)× 107 copies/μg RNA and (8.49±0.67)×105 copies/μg RNA, respectively. The former was 80.8 times greater than the latter. Each sample was measured 10 times and the coefficient variation (CV) was 9.5% and 7.9%, respectively. Various levels of MDR1 gene expression were detected in 12 of 45 patients with lung cancer. Conclusion: Quantitative detection of MDR1 gene expression in tumor cells was achieved by using FQ-RT-PCR. FQ-RT-PCR is an accurate, and sensitive method and easy to perform. Using this method, low levels of MDR1 gene expression could be detected in 24% of the patients with lung cancer.

Establishment of a Real-time Fluorescent Quantitative RTPCR Method for Detecting NP Gene of Class Ⅰ Newcastle Disease Virus(NDV)

Newcastle disease( ND) is one of the most serious infectious diseases that infect the poultry industry.There is only one serotype of Newcastle disease virus( NDV),but NDVs can be divided into two distinct classes( class Ⅰ,and class Ⅱ) according to their genetic relationship.To develop a method for rapid quantitative detection of class Ⅰ NDV,a pair of primers and a TaqM an probe were designed and synthesized according to the conservative sequence of NP gene of class Ⅰ NDV.The positive recombinant plasmid harboring NP gene of JS-18-05 isolate was used as a positive template to establish the standard curve.A real-time fluorescent quantitative RT-PCR method was established for rapid detection of class Ⅰ NDV with strong specificity,high sensitivity and good repeatability.The established method exhibited a good linear relationship within the concentration of 102 to 108 copies of NDV,by which 1 μl of 10 copy of NDV nucleic acid could be detected in the initial template.Compared with conventional virus isolation methods,the established method had similar sensitivity and led to the same results in detecting33 class Ⅰ,class Ⅱ NDV isolates.The study provided the basis for rapid quantitative detection of class Ⅰ NDVs and further clarification of their pathogenicity and pathogenic mechanism in poultry.

Method for Solving Non-specific Amplification Interference of Fluorescence Quantitative PCR in Gene Detection

Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.

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%.

Development and Preliminary Application of SYBR Green I Real-Time Fluorescence Quantitative PCR Method for Detecting Porcine Parvovirus Virus

According to VP2 gene sequence of the porcine parvovirus virus strain NADL-2 （NC001718） available in GenBank （NC_001718）, a pair of specific primer was designed, and the target fragment of 431 bp was obtained by PCR amplification. The products were ligated with pMD18- T vector and then transformed into bacteria DH5α for recombinant plasmid extraction. After PCR identification and sequencing, recombinant plasmid was used as a standard template to establish the standard curve of SYBR Green I fluorescence quantitative PCR. Sensitivity test, specificity test and repeatability test were also determined. The results indicated that there was a good linear relationship between threshold cycle of the standard curve and template concentration, R2 =0.997 6. Tm ranged from 82.3 to 82.9 ℃, while the sensitivity was 72.1 copies/μl with good specificity and repeatability. The developed SYBR Green I real-time quantitative PCR method to detect PPV VP2 gene laid the basis for further studies on patho- oenesis, early clinical diaonosis of this virus and quantitative analysis of PPV infection.

基于盖他病毒nsP3基因的TaqMan荧光定量RT-PCR检测方法的建立及初步应用

为建立一种灵敏、特异且高效的检测盖他病毒(GETV)的TaqMan荧光定量RT-PCR方法,本研究根据GETV的nsP3基因设计特异性引物及探针,以GETV (SC483株) cDNA作为模板,扩增目的基因并克隆至p ET-29a载体中,构建重组质粒标准品p ET29a-nsP3并经PCR和测序鉴定。基于该质粒标准品,采用方阵法优化引物、探针浓度以及退火温度,初步建立了一种基于GETV nsP3基因的TaqMan荧光定量RT-PCR检测方法,并绘制标准曲线。以GETV (SC483株、SC266株)、辛德毕斯病毒(SINV)、猪流感病毒(H3N2、SIV)、猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)、猪繁殖和呼吸障碍综合征病毒(PRRSV)等病毒的基因组RNA反转录为cDNA作为模板,利用本实验建立的TaqMan荧光定量RT-PCR方法检测,结果显示,仅GETV为阳性,SINV、SIV、TGEV、PEDV、PRRSV均为阴性,表明该方法特异性较强;分别以10倍倍比稀释(3.07×10^(-1)拷贝/μL~3.07×10^(8)拷贝/μL)的重组质粒标准品p ET29a-nsP3作为模板,利用本研究建立的方法和普通RT-PCR方法分别检测,结果显示,本研究建立的方法对重组质粒标准品的检测限为30.7拷贝/μL,敏感性是普通RT-PCR的100倍,敏感性较高;分别以同一时间及不同时间提取的不同浓度的质粒标准品作为模板,利用本研究建立的荧光定量RT-PCR进行组内与组间的重复性试验检测,结果显示,该方法的组内组间变异系数均小于2%,重复性较好。利用本实验建立的TaqMan荧光定量RT-PCR方法与普通RT-PCR方法分别对来源于GETV感染小鼠的84份组织样品中的病毒核酸进行检测,结果显示,该TaqMan荧光定量RT-PCR方法对GETV检出率为95.24%(80/84),而普通RT-PCR对GETV的检出率为67.86%(57/84)。二者的阳性符合率达100%。本研究首次基于GETV nsP3基因建立了TaqMan荧光定量RT-PCR检测方法,该方法特异性强、敏感性高、重复性好,为GETV的检测提供了有效工具。

Development of real-time PCR method for rapid detection and quantification of Heterosigma akashiwo

To rapidly detect the harmful algae H.akashiwo qualitatively and quantitatively, sequences of the 18S rDNA deduced from H.akashiwo were used for designing species-specific primers, and a RFQ-PCR (Real-time Fluorescent Quantitative Polymerase Chain Reaction) method was developed for quantitative detection of H.akashiwo. Primer H.akashiwo and TaqMan probe were designed, and the specificity of primer was checked with PCR. A calibration curve was constructed with cycle threshold value against visual counted cell number. And the value of the curve was tested with other H.akashiwo samples, which were assayed with both the RFQ-PCR method and visual count under microscope.

Taqman PCR-based Methods for Rapid Detection of Salmonella in Pet Food

[ Objective] To establish a Taqman real-time PCR for detection of Salmonella in pet food. [Method] A pair of primers and a probe were designed based on published nucleotide sequence of invA gene encoding the invasion protein of Salmonella enterica. [ Result] The assay detects Salmonella specifically. The detection limit of the real-time PCR was 17 CFU/test （25 uL/test） for the positive strain. This method was effective to detect artificially contaminated pet food. [ Conclusion] The results showed that Taqman PCR assay was rapid and accurate for detection of Salmonella from infected pet food.

塞内卡病毒实时荧光定量RT-PCR检测方法的建立及应用

为加强塞内卡病毒(SVA)在不同宿主的诊断及流行病学监测与防控,根据GenBank中发表的SVA VP1基因区域设计一对特异性引物和带有TaqMan发光基团标记的探针,构建重组阳性质粒并用作建立实时荧光定量RT-PCR方法的模板,对反应体系和条件进行优化,构建标准曲线并验证该方法的特异性、敏感性和重复性。结果显示,该方法的最适退火温度为55℃,最佳引物和探针浓度分别为0.2μmol/L和0.4μmol/L,最佳反应循环数为45,Ct值与标准品浓度的对数线性关系好。对猪源、鼠源及牛源SVA和其他动物病毒(O型口蹄疫病毒、水疱性口炎病毒、牛病毒性腹泻病毒、猪伪狂犬病病毒、边界病毒、猪库布病毒、牛库布病毒、山羊鼻内肿瘤病毒、牛传染性鼻气管炎病毒、牛结节性皮肤病病毒、牛冠状病毒、D型流感病毒、蓝舌病毒)的核酸检测结果显示,除猪源、鼠源及牛源SVA为阳性外,其他病毒核酸为阴性。该方法最低检测限为2.66×10~1拷贝/μL,重复试验中组内和组间变异系数均小于0.1%。建立的检测多宿主SVA TaqMan RT-qPCR方法具有良好的特异性和稳定性,为SVA在不同宿主间的疾病诊断与流行病学调查或监控提供了快捷的方法。

RAPID DETECTION OF ONCOGENE AMPLIFICATION IN PARAFFIN SECTIONS OF BREAST CARCINOMAS USING QUANTITATIVE DIFFERENTIAL PCR AND FLUORESCENT DNA TECHNOLOGY

Gene amplification is a common mechanism of oncogene activation and contributes to tumor progression. Analysis of such genetic alterations are relevant to the understanding of tumor genetics and could provide prognostic information for the individual patient. Standard analytical approaches using Southern blot and slot blot require a large amount of good

BVDV-1型实时荧光定量RT-PCR检测方法的建立及应用

【目的】建立特异针对牛病毒性腹泻病毒基因1型(BVDV-1)的实时荧光定量RT-PCR检测方法。【方法】通过比对分析近5年流行的BVDV-1毒株全基因序列,基于保守序列5′-UTR设计特异性引物和探针,优化反应体系和反应条件,并进行特异性、敏感性、重复性及临床样品检测试验。【结果】BVDV-1检测的最佳探针浓度为0.25μmol/L,引物浓度为0.60μmol/L,对阳性质粒标准品的最低检测限为1.7 copies/μL。特异性试验显示:建立的方法仅能特异性扩增BVDV-1,对蓝舌病、口蹄疫、水泡性口炎、小反刍兽疫和猪瘟等病毒不发生交叉反应,特异性良好,变异系数小于1%。采用建立的方法对172份临床血液样本进行BVDV核酸检测,结果检出BVDV阳性样本33份,阳性率为19.19%,与《牛病毒性腹泻/粘膜病诊断技术规范》(GB/T 18637—2018)中实时荧光定量RT-PCR检测结果一致。【结论】本研究建立的BVDV基因1型实时荧光定量RT-PCR检测方法特异性强、敏感性高、重复性好,可用于临床样本的检测,为BVDV-1的检测及监测提供了有力的技术手段。

Rare-earth ions coordination enhanced ratiometric fluorescent sensing platform for quantitative visual analysis of antibiotic residues in real samples

Levofloxacin(LVFX)as a representative drug of quinolone antibiotics is widely used in clinical,and its residues enriched in water bodies and sideline products seriously damage human health.It is imperative to develop a real-time/on-site sensing method for monitoring residual antibiotics.Here,we report a portable sensing platform by utilizing a composite fluorescent nanoprobe constructed by the cerium ions(Ce^(3+))coordination functionalized Cd Te quantum dots(QDs)for the visual and quantitative detection of LVFX residues.This fluorescent probe provides a distinct color variation from red to green,which shows a good linear relationship to LVFX residues concentrations in the range of 0-6.0μmol/L with a sensitive limit of detection(LOD)of 16.3 nmol/L.The smartphone platform with Color Analyzer App installed,which could accomplish quantified detection of LVFX in water,milk,and raw pork with a LOD of 27.9nmol/L.The facile sensing method we proposed realizes rapid visualization of antibiotics residual in the environment and provides a practical application pathway in food safety and human health.

牛冠状病毒TaqMan荧光定量PCR检测方法的建立及应用

为建立牛冠状病毒TaqMan荧光定量PCR检测方法,根据GenBank收录的牛冠状病毒AKS-01株N基因序列(KU886219)保守区设计特异性引物和探针,构建重组质粒并进行反应条件优化、特异性试验、重复性试验以及敏感性试验,建立一种检测BCoV的TaqMan荧光定量PCR方法。结果显示,建立的牛冠状病毒TaqMan荧光定量PCR检测方法特异性、敏感性和重复性均良好。该方法BCoV重组质粒标准品在5.75×10^(7)~5.75×10^(3)copies/μL时与Ct值呈现良好线性关系,该方法对牛轮状病毒、牛传染性鼻气管炎病毒、牛病毒性腹泻病毒、牛副流感病毒3型均无交叉反应,特异性良好;该方法对BCoV重组质粒标准品最低检测限为5.75×10^(1)copies/μL;批内和批间重复性试验结果稳定,变异系数均小于2%。利用所建立的TaqMan荧光定量PCR方法对收集的132份样品进行检测,与常规PCR相比,两者符合率为96.21%,可为BCoV的临床检测和流行病学调查提供技术支持。

水禽细小病毒SYBR Green Ⅰ荧光定量PCR检测方法的建立与应用

为了建立水禽细小病毒(WPV)快速检测方法,根据序列比对结果在水禽细小病毒NS基因SF3保守区域内设计特异性引物,建立SYBR Green Ⅰ荧光定量PCR通用检测方法。该方法的扩增效率(E)为90.0%,相关系数(R~2)=0.99,标准曲线方程为y=-3.607x+38.77;除WPV出现S形扩增曲线外,新城疫病毒(NDV)、H9亚型禽流感病毒(H9 AIV)、鸭坦布苏病毒(DTMUV)、鸭肝炎病毒(DHAV)、鸭肠炎病毒(DEV)、鸭呼肠孤病毒(DRV)样品均未出现S形阳性扩增曲线;批内变异系数(CV)为0.15%~0.23%,批间变异系数为0.09%~0.28%。结果表明,SYBR Green Ⅰ荧光定量PCR检测方法重复性好、灵敏度高和特异性强。临床样品检测结果表明,SYBR Green Ⅰ荧光定量PCR与普通PCR的符合率达98.4%,灵敏度是普通PCR的1 000倍。SYBR Green Ⅰ荧光定量PCR检测方法不仅能定性检测WPV,还可以进行定量检测,可用于种鸭场、种鹅场的WPV净化检测,也可用于WPV临床大量样品的快速检测。