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 samples. 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.

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) polymerase 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 compared with that of conventional PCR. [Result] The sensitivity of fluorescence quantitative PCR was higher than that of conventional PCR. The minimum copy number that could be detected was 1.6×102 copies/μl. 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.

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 pathogenesis,early clinical diagnosis of this virus and quantitative analysis of PPV infection.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Manila Clam Ruditapes philippinarum Under Hypoxic Stress

Quantitative real-time PCR(qRT-PCR)has been widely used for gene expression analysis,and selection of reference genes is a key point to obtain accurate results.To find out optimal reference genes for qRT-PCR in Manila clam Ruditapes philippinarum in response to hypoxia,different tissues were used and compared to evaluate the stability of candidate reference genes under low oxygen stress(DO 0.5mgL^(−1) and DO 2.0mgL^(−1))and normal condition(DO 7.5mgL^(−1)).Seven candidate reference genes were selected to evaluate the stability of their expression levels.The reference genes were evaluated by Delta Ct,BestKeeper,NormFinder and geNorm,and then screened by RefFinder calculation.Under hypoxic stress of 0.5mgL^(−1),the most suitable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were TUB and HIS,respectively.For hypoxic stress of 2.0mgL^(−1),the most stable reference gene for gill and hepatopancreas was RPL31,and the optimal reference genes for axe foot and adductor muscle were RPS23 and EF1A,respectively.At the normal condition,HIS and EF1A were identified as the optimal internal reference genes in gill and hepatopancreas respectively,and GFRP2 was the best internal reference gene for axe foot and adductor muscle.The present findings will provide important basis for the selection of reference genes for qRT-PCR analysis of gene expression level in bivalves under hypoxic stress,which might be helpful for the analysis of other molluscs too.

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 generated typical amplification curves with high amplification efficiencies(E = 1. 06,1. 07,0. 92 and 0. 92,respectively),good linear 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 real-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<50×109 group(P > 0.05). In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD>103 group than that in MRD<103 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 marrow 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.

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。

Evaluation of Clinical Application of Chemiluminescence and Real-time,Fluorescence-based Quantitative PCR in Diagnosis of Epstein-Barr Virus lnfection

Objective:To compare the effects of clinical application of chemiluminescence and real-time,fluorescence-based quantitative PCR in the detection Epstein-Barr virus(EBV).Methods:The data of chemiluminescence and real-time fluorescent quantitative PCR.fromipaEsfwo were suspected of being infectea w1tn rito1 roro January 2016 to January 2019 in our hospital were analyzed.The specific stage of EBV infection was analyzed,and the differences in results of the two detection methods were compared.Results:Chemiluminescence method was used to detect EBV infection during the active phase.The sensitivity of the chemiluminescence method was 76.7%(56/73)and the real-time quantitative PCRmethod was 90.4%(66/73).There was a statistical difference between the two detection methods(P<0.05).Conclusion:There was no statistical difference in positive predictive values between the chemiluminescence method and the real-time,fluorescence-based quantitative PCR method in the detection of EBV infection,but the sensitivity of chemiluminescence method is slightly lower than the real-time quantitative PCRmethod.It is noteworthy that chemiluminescence method is convenient and fast while the real-time,fluorescence-based quantitative PCR method is more accurate,which can provide a more accurate reference for clinical treatment.

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 16 S rRNA gene sequences of common Lactobacillus species in fermented material. Bacterial genome DNA of separated L. acidophilus in fermented sample was taken as template,and L. acidophilus in fermented material was conducted the quantitative determination by real-time quantitative PCR( RT-PCR). Analysis on RT-PCR results shown that contents of L. acidophilus 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 applied 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.

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

Real-time 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 disadvantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the application and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.

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 replications.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 4;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 organisms 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 amplified gene was cloned into the vector of pMD~18-T and transformed into DH5α to screen positive clones.After being extracted and purified,the recombinant plasmids 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 reaction 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 3.21×10~0-4.16×10~8 copies /μL,the correlation coefficient was 0.998 8 and the slope was-3.286.The method did not show any cross-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 trials 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 real-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established,which was better for conducting the quantitative analysis and the early diagnosis of PCV2 infection.

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.

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.

Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction

Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.

Effects of Different Temperature and Time Durations of Virus Inactivation on Results of Real-time Fluorescence PCR Testing of COVID-19 Viruses

Summary:The novel coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan,Hubei province of China in January 2020.This study aims to investigate the effects of different temperature and time durations of virus inactivation on the results of PCR testing for SARS-CoV-2.Twelve patients at the Renmin Hospital of Wuhan University suspected of being infected with SARS-CoV-2 were selected on February 13,2020 and throat swabs were taken.The swabs were stored at room tempcrature(20-25℃),then divided into aliquots and subjected to different temperature for different periods in order to inactivate the viruses(56℃for 30,45,60 min;65,70,80℃for 10,15,20 min).Control aliquots were stored at room temperature for 60 min.Then all aliquots were tested in a real-time fluorescence PCR using primers against SARS-CoV-2.Regardless of inactivation temperature and time,7 of 12 cases(58.3%)tested were positive for SARS-CoV-2 by PCR,and cycle threshold values were similar.These results suggest that virus inactivation parameters exert minimal infuence on PCR test results.Inactivation at 65℃for 10 min may be sufficient to ensure safe,reliable testing.

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.