Detection of Ratoon Stunting Disease in Virus-free Seedcane via Real-time Fluorescence Quantitative PCR

This study was to develop the real-time fluorescence quantitative PCR technique for detecting the ratoon stunting disease （RSD） in virus-free seedcane seedlings. Healthy tissue culture seedlings were obtained from six plants of sugarcane ROC22, which had been confirmed RSD-positive by detecting the sugarcane juice, by employing the sugarcane seedlings production protocol. Real-time fluorescence quantitative PCR was used to detect RSD pathogens in tissue culture sam- pies. The results showed that target fragment of RSD pathogens was not found in all 10 samples in real-time fluorescence quantitative PCR, with the Ct values of 37 - 39. The healthy tissue culture sugarcane seedlings do not carry RSD pathogens, indicating that adopting healthy seedcane seedlings production technique could thoroughly get rid of RSD pathogens.

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.

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.

Development of a Fluorescence Quantitative PCR Method for Detection of Marteilia refringens in Shellfish

[Objective] This paper was to develop a fluorescence quantitative PCR method for detection of M. refringens in shellfish. [Method] A pair of primers and a TaqMan probe were designed and synthesized according to the conserved gene sequences of M. refringens in GenBank, so as to develop a fluorescence quantitative PCR method for detection of M. refringens. The developed fluorescence quantitative PCR method was compared with conventional PCR detection. [Result] The fluorescence quantitative PCR could detect 40 template copies of plasmid DNA, and its sensitivity was 100 times higher than the conventional PCR. The detection results of Perkinsus sp, Haplosporidium sp, Aeromonas hydrophila, Pseudomonas fluorescens, Vibrio parahaemolyticu, Vibrio alginolyticu, Vibrio rluvialis and Vibrio mimicus were negtive. [Conclusion] The fluorescence quantitative PCR method for M. refringens established in this paper is specific, sensitive, rapid and quantitative with good repeatability, which can be used for clinical detection of M. refringens infection.

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.

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 Application of Realtime Fluorescence Quantitative PCR for Prenatal Screening of Group B Streptococcal Infections

Objective: In the prenatal screening, several different methods were used to detect the presence of group B streptococcus (GBS) infection, in this assay, the diagnostic value and clinical significance of the application of realtime fluorescent PCR were explored. Methods: A total of 86 women with 35-37 weeks pregnancy were enrolled, vaginal secretion samples were collected. Fluorescence PCR, bacterial culture and gene sequencing were used to detect whether there was GBS infection, and the results obtained were compared and analyzed. Results: 10 subjects were detected to be positive for GBS by fluorescence PCR (the positive rate was 11.6%), however, only 4 cases were positive for GBS by bacterial culture method (the positive rate was 4.7%). There was a statistically significant difference in the positive rate between the two methods (P<0.01). Compared with the results of gene sequencing, the detection of GBS infection by fluorescence PCR has an accuracy of 95.2%, and the sensitivity was 90.9% with 100% specificity. Conclusion: The application of realtime fluorescence quantitative PCR for the detection of GBS infection is significantly better than the use of bacterial culture method. Compared with the gold standard method (gene sequencing method), its detection efficiency, accuracy, sensitivity and specificity are relatively high. In summary, PCR for prenatal screening of GBS is worthy of promotion in clinical practice.

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.

Real-time fluorescent quantitative PCR非特异性扩增的研究进展

Real-time fluorescent quantitative PCR(RT-qPCR)因其高效快速、操作简便、高度敏感等优点获得广泛应用,但因其强大的放大功能而容易出现非特异性扩增的问题,尤其是荧光染料法中更加明显。文章对RT-qPCR的原理、非特异性扩增的发生因素、解决方案及该技术的应用前景进行了综述。

Application of Real-time Fluorescent Quantitative PCR in Plant

Real-time fluorescent quantitative PCR （RQ-PCR） is a detection method by adding fluorescent dye or fluorescent probe into the PCR reaction system, using fluorescent signal accumulation to monitor amplification reactions of PCR reaction process, and finally the unknown template can be quantitatively analyzed through the standard curve. So the detection level of PCR has improved from the qualitative to the quantitative. In order to provide a theoretical reference for further application, the principle, classification, advantages and disadvantages of RQ-PCR were intro- duced, and its application and progress in plants in recent years were reviewed.

SAT-TB联合FQ-PCR检测在痰涂片阴性肺结核诊断中的效能

目的:分析RNA恒温扩增实时检测技术(SAT-TB)联合荧光定量聚合酶链反应(FQ-PCR)检测在痰涂片阴性肺结核患者诊断中的效能。方法:选取2020年7月至2022年7月该院收治的75例疑似痰涂片阴性肺结核患者进行前瞻性研究。采集所有患者肺泡灌洗液标本,采用SAT-TB、FQ-PCR法进行检测,以结核分枝杆菌痰培养结果为“金标准”,比较SAT-TB、FQ-PCR单项及联合检测在痰涂片阴性肺结核诊断中的效能。结果:金标准结果显示,75例疑似痰涂片阴性肺结核患者中,阳性48例,阴性27例;SAT-TB检测结果显示,阳性36例,阴性39例;FQ-PCR检测结果显示,阳性37例,阴性38例;SAT-TB联合FQ-PCR检测结果显示,阳性47例,阴性28例;SAT-TB联合FQ-PCR检测诊断痰涂片阴性肺结核的灵敏度、准确度均高于SAT-TB、FQ-PCR单项检测诊断,漏诊率低于SAT-TB、FQ-PCR单项检测诊断,差异有统计学意义(P<0.05)。结论:SAT-TB联合FQ-PCR检测诊断痰涂片阴性肺结核的效能高于二者单项检测诊断效能。

Application of Real-time Fluorescent Quantitative PCR in Studies on Plants

Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.

Detection of Lactobacillus acidophilus in Fermented Material by Real-time Fluorescent Quantitative PCR

The species distinctive PCR primer of Lactobacillus acidophilus （ L. acidophilus） was designed according to 16S rRNA gene sequences of conunon Lac- tobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template, and L. acidophilus in fer- mented material was conducted the quantitative determination by real-time quantitative PCR （RT-PCR）. Analysis on RT-PCR results shown that contents of L. aci- dophilus in the test sample reached 1.5 billion CFU / g. Test results shown that contents of L. acidophilus in fermented material could be detected accurately by the established RT-PCR method in the test. indicating that the established RT-PCR method could be aookued to the detection of L. acidophilus in fermented material.

Real-time fluorescent quantitative immuno-PCR method for determination of fluoranthene in water samples with a molecular beacon

A reliable and sensitive competitive real-time fluorescent quantitative immuno-PCR （RTFQ-IPCR） assay using a molecular beacon was developed for the determination of trace fluoranthene （FL） in the environment.Under optimized assay conditions,FL can be determined in the concentration range from 1 fg/mL to 100 ng/mL,with y=0.194x ＋ 7.859,and a correlation coefficient of 0.967 was identified,with a detection limit of 0.6 fg/mL.Environmental water samples were successfully analyzed,recovery was between 90% and 116%,with intra-day relative standard deviation （RSD） of 6.7%-12.8% and inter-day RSD of 8.4%-15.2%.The results obtained from RTFQ-IPCR were confirmed by ELISA,showing good accuracy and suitability to analyze FL in field samples.As a highly sensitive method,the molecular beacon-based RTFQ-IPCR is acceptable and promising for providing reliable test results to make environmental decisions.

Establishment of a Real-time Fluorescent Quantitative PCR Assay for Detection of Genetically Modified Maize Line MON88017

In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms （GMOs） and products, ensure bio-safety and reduce ecological risk in China, a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017. The established method was evaluated based on the specificity, sensitivity, accuracy and measurement uncertainty. The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017. 1.50% MON88017 sample was detected with 29 replica- tions. The average measured value （ 1. 541% ） was close to the actual value （ 1.50% ） and the relative deviation was 2.70%. The variation coefficient of the measured value was 0.110 g ; the recovery was 100.00% and the measurement uncertainty was 0. 096. The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5% confidence level. Thus, the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity, accuracy and sensitivity, which could provide technical support for the safety supervision of genetically modified organ- isms and products in China.

Establishment and Application of a Real-time Fluorescent Quantitative PCR Method for Detection of Porcine Circovirus Type 2

[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction （PCR） method with SYBR Green I for the detection of porcine circovirus type 2 （PCV2）. [Methods] Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR. The ampli- fied gene was cloned into the vector of pMD 18-T and transformed into DHSct to screen positive clones. After being extracted and purified, the recombinant plasraids pMD 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2, and the PCR re- action conditions were optimized. [ Results] Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21 × 100 -4.16 × 108 copies/μL , the correlation coefficient was O. 998 8 and the slope was - 3.286. The method did not show any cress-reactions with the genomes of PRRSV, PCV1, CSFV, PRV, PPV and Escherichia coli. Sensitivity of this method was proved to be 3.21 × 10 copies/μL, which was 1 000 times higher as conventional PCR method. Variation coefficients of the repeated trims among same batch or different batches were both less than 3.00%. Positive rate of clinical samples detected by the established PCR method was 58.94%, which was significantly higher than the detection rate by conventional PCR. [ Conclusions ] A reM-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established, which was better for conducting the quan- titative analysis and the early diagnosis of PCV2 infection.

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.

Detection of the covalently closed circular DNA of duck hepatitis B virus by Taq-Man fluorescent quantitative PCR assay

To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA （eccDNA） of duck hepatitis B virus （DHBV）, a pair of primers was designed from both sides of the nick in the minus strand of DHBV and a Taq-Man probes between the primers, modified with 6-Fam at 5＇ end and Tamra at its 3＇ end was designed to detect the PCR products during PCR cycles. The DHBV DNA fragment was cloned into vector PUCm-T, and the recombinant plasmid was purified and subsequently qualified as the HBV DNA standard. The experimental conditions and reagents used in PCR assay for amplification were sophisticatedly optimized in order to yield a perfect amplification efficacy and reduce the possibility to produce non-specific amplification. It was demonstrated that the detect limit of assay was 10^3 copies/ml, and a linear standard curve was obtained between 10^5 -10^9 copies/ml [ C1 =-2.8361 ln（x） ＋ 41.45, r =-0.9985]. The coefficient of variation was 0.2%-3.14% and 2.22%-4.43% for intra- and inter-assay respectively. After a dynamic survey on the contents of DHBV DNA in serum of ducks, it was found that its peak value appeared at the second week of birth in ducks. It is evident that this method of Taq-Man fluorescent quantitative PCR assay appears to be simple, sensitive and specific.

5种猪源消化道传播病原体荧光定量PCR检测方法的建立

猪源人兽共患病不仅威胁公共卫生安全,还威胁生猪养殖业的健康发展;其中消化道传播是其主要的传播途径。为及时鉴定出经消化道传播的病原体,本试验建立了猪源大肠杆菌、沙门菌、单增李斯特菌和志贺菌的四重荧光定量PCR和戊型肝炎病毒荧光定量PCR检测方法,并优化反应体系和条件,实现5种病原体的同时检测;并利用荧光定量PCR与普通PCR对117份猪临床样品(病变组织、粪便和肌肉等)进行对比检测。结果显示,建立的荧光定量PCR能够在1.5 h内完成对大肠杆菌、沙门菌、单增李斯特菌、志贺菌和戊型肝炎病毒5种病原体的特异性检测,与其他常见细菌和病毒无交叉反应,检测极限值可达5个拷贝,标准曲线相关系数均不低于0.997,线性范围涵盖1×10^(1)~1×10^(9),批内和批间变异系数(CV)均低于3.16%。建立的荧光定量PCR与普通PCR方法检测结果的符合率达到95.73%~100%,具有较好的一致性。117份临床样品中大肠杆菌、沙门菌、单增李斯特菌、志贺菌和戊型肝炎病毒各自的阳性率分别为31.62%、17.95%、6.84%、5.13%和11.11%。结果表明,本试验所建立的荧光定量PCR方法灵敏、特异、稳定,能够同时、快速区分检测上述5种猪源消化道传播病原体,可为猪肉制品从产地到餐桌全环节样品的监测提供有力的技术支持。