EVALUATION OF TWO REAL-TIME PCR METHODS FOR DETECTION OF HPdel, A RISK MUTATION FOR ANAPHYLACTIC SHOCK, BEFORE TRANSFUSION
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Abstract:
重篤な非溶血性輸血後副作用であるアナフィラキシーショックのリスク因子であるハプトグロビン欠損症の原因遺伝子はハプトグロビン遺伝子欠失（HPdel）であり，その頻度は約4,000人に1人であると予想される．我々は最近安全な輸血医療の遂行を目的とし迅速・簡便なリアルタイムPCR法に基づく2種のHPdel診断法を開発し報告した．今回，臨床現場への導入を目的として久留米大学病院で輸血予定患者の血液を鋳型とし，TaqMan probeを用いる方法とSYBR green Iを用い融解曲線をおこなう方法の2法を実施し結果を比較した．約1時間半で得られた結果は全サンプルで一致し，2009年1月から2010年3月末に解析した2,954名のうち91名がHP/HPdel，1名がHPdel/HPdelであった．TaqMan法は増幅シグナルそのものが結果を反映することから反応中に診断結果を予想でき，HPdel/HPdelがソフトウエアで自動検出できるため多検体処理能力に優れた方法であり，SYBR法は初期費用が低く抑えられ，より幅広い臨床現場での導入が容易であると考えられた．Keywords:
TaqMan
Anaphylactic shock
SYBR Green I
Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×10 7 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity.In this study, the plate count method was performed using surface-plating of 0.1 ml of each sample on Palcam Agar.The lowest detectable level for this method was 1.58×10 cfu/ml for both nutrient broth and milk samples.Using purified DNA as a template for generation of standard curves, as few as four copies of the iap-gene could be detected per reaction with both real-time PCR assays, indicating that they were highly sensitive.When these real-time PCR assays were applied to quantification of L. monocytogenes in decimal serial dilution series of nutrient broth and milk samples, 3.16×10 to 3.16×10 5 copies per reaction (equals to 1.58×10 3 to 1.58×10 7 cfu/ml L. monocytogenes) were detectable.As logarithmic cycles, for Plate Count and both molecular assays, the quantitative results of the detectable steps were similar to the inoculation levels.
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SYBR Green I
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Real‐time quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) is the method of choice for rapid and reproducible measurements of cytokine or growth factor expression in small samples. Fluorescence detection methods for monitoring real‐time PCR include fluorogenic probes labelled with reporter and quencher dyes, such as Taqman probes or Molecular Beacons and the dsDNA‐binding dye SYBR Green I. Fluorogenic (Taqman) probes for a range of human and rat cytokines and growth factors were tested for sensitivity and compared with an assay for SYBR Green I quantification using real‐time fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detector). SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. Fluorogenic probes provided sensitive and reproducible detection of targets that ranged from low (<10 copies/reaction) to high (>10 7 copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (≥1000 copies/reaction), but did not give consistently reproducible quantification when the target gene was expressed at low levels. Although optimization of melting temperature improved the specificity of SYBR Green I detection, in our hands it did not equal the reproducible sensitivity and specificity of fluorogenic probes. The latter method is the first choice for measurement of low‐level gene expression, although SYBR Green I is a simple and reproducible means to quantify genes that are expressed at moderate to high levels.
TaqMan
SYBR Green I
Molecular beacon
Primer dimer
Melting curve analysis
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Objective To establish a real-time quantitative RT-PCR method for detection of HLA-DRα mRNA expression.Methods The vector containing HLA-DRα gene as standard template was constructed by T-A cloning technique.A real-time RT-PCR was established by used the fluorogenic probe(Taqman probe).HLA-DRα mRNA expression level in monocytes induced by achyrathes bidentata polysacchari-(des(ABPS)) was detected with real-time quantitative RT-PCR and semi-quantitative RT-PCR.Results The expression of HLA-DRα mRNA in monocytes induced by ABPS was increased significantly,but no significant change of HLA-DRα mRNA expression was fund in control group;The semi-quantitative RTPCR results also demonstrated the variety of HLA-DRα level as what the real-time fluorogenic quantitative RT-PCR detected,but less sensitive and accurate.Conclusion The fluorogenic real-time quantitative RT-PCR is more accurate and sensitive than semi-quantitive RT-PCR in Detection of HLA-DRα mRNA expression.
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Quantitative Analysis
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Objective To establish a real-time quantitative RT-PCR method for the detection of expression of TNF-α mRNA.Methods The vector containing TNF-α gene as standard template was constructed with T-A cloning technique.The fluorogenic probe(i.e,Taqman probe)was used to establish a real-time RT-PCR.TNF-α mRNA expression level in human peripheral blood mononuclear cells(PBMC) for tumor patients and normal persons was determined with real-time quantitative RT-PCR,also with semi-quantitative RT-PCR.Results The expression of TNF-α mRNA in PBMC for tumor patients was more high than that of normal persons;The semi-quantitative RT-PCR results also demonstrated that TNF-α level varied as detected with real-time fluorogenic quantitative RT-PCR,but less sensitive and accurate.Conclusion Detection of TNF-α mRNA expression with the fluorogenic real-time quantitative RT-PCR is more accurate and sensitive than semi-quantitive RT-PCR.
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Quantitative Analysis
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Objective To compare the sensitivity, specificity and time-spending of TaqMan real-time RT-PCR and conventional RT-PCR for detecting dengue virus. Methods The real-time RT-PCR and the conventional RT-PCR assays were used to quantitatively detect dengue virus, Japanese encephalitis virus and West Nile virus and the sensitivity, specificity and time-spending were evaluated between the two methods above. Results Both Japanese encephalitis virus and West Nile were not detected by those two methods, however, dengue virus could be detected by both two methods. The sensitivity of the conventional RT-PCR was 0.1 TCID50/mL, while TaqMan real-time quantitative RT-PCR was 0.001TCID50/mL, which was 100 times sensitive than conventional RT-PCR. Meanwhile, the real-time RT-PCR was more time-saving than conventional RT-PCR. Conclusion TaqMan quantitative real-time RT-PCR assay is more sensitive and time-saving than conventional RT-PCR for the rapid diagnosis of dengue virus.
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Quantitative RT-PCR using the TaqMan® Gene Expression Assays on StepOnePlus™ Real-Time PCR System v1
Quantitative RT-PCR using the TaqMan® Gene Expression Assays on StepOnePlus™ Real-Time PCR System
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TaqMan
SYBR Green I
Amplicon
SDHA
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Objective To explore the improvement of detecting HBV-DNA by fluorescent quantitative polymerase chain reaction(FQ-PCR)using both SYBR green I and TaqMan probe in the reaction.Method 3 HBV-DNA positive serum samples(108.48,105.70 and 103.70 copies/ml)and 1 HBV-DNA negative serum sample were used to detect HBV-DNA by the double fluorescent quantitative PCR(TaqMan+SYBR Green I group),SYBR Green I PCR(SYBR Green I group)and TaqMan probe PCR(TaqMan goup).The detection of the HBV-DNA of each sample was performed for 5 times.Result The HBV-DNA concentrations of the 3 HBV-DNA positive serum samples detected by TaqMan+SYBR Green I PCR were 108.55±0.32,105.79±0.29 and 103.81±0.30,while those detected by TaqMan PCR were 108.49±0.31,105.69±0.30,103.72±0.25 copies/ml.There was no statistically significance between two methods(t=0.31,0.54 and 0.27,resectively P 0.05).The HBV-DNA concentration detected by SYBR Green I PCR of the 3 positive samples were 108.41±0.35,105.21±0.34 and 103.26±0.26 copies/ml.The sensitivity of SYBR Green PCR in detecting HBV-DNA showed no significant difference to that of TaqMan+SYBR Green I PCR for the sample with a high HBV-DNA copy number(108.48 copies/ml)(t=0.68,P0.05),but was significantly lower for the samples with lower HBV-DNA copy number(105.70 and 103.70 copies/ml)(t=2.90,2.62,respectively P0.05).The melting curves were detected in both TaqMan+SYBR Green I group and SYBR Green I group,with Tm of 71.8℃,72℃ and 79.8℃,but was not detected in HBV-DNA negative serum samples.Conclusion Sensitivity and specificity of detecting HBV-DNA by SYBR Green I combined with TaqMan PCR are higher than only using SYBR Green I or TaqMan probe.
TaqMan
SYBR Green I
Primer dimer
genomic DNA
Taq polymerase
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To search a novel sensitive, specific and lower cost method applicable for quantitative analysis of the hepatitis B virus DNA extensively.Quantitative analysis of the DNA from 100 sera by real-time PCR with Sybr green 1. The results of Sybr's assay were compared with the results obtained with Taqman's fluorescent quantitative assay.Taqman real-time PCR could help evaluate the level of virus reliably. The results of Sybr's assay were in agreement with the Taqman's assay, but detection rate was lower.Sybr green 1 real-time PCR appeared to be convenient and cheap, but detection rate was lower.
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SYBR Green I
Primer dimer
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The probes and primers were designed and synthesized according to the conserved gene ORF7 of PRRSV available in GenBank,and then reaction parameters were optimized to develop a real-time TaqMan-quantitative RT-PCR assay.The serum and other tissue samples from pigs on farms in Dongguan,Zengcheng,Zhanjiang and other areas of Guangdong Province were detected by using the established quantitative RT-PCR assay,and the results was compared with that of routine RT-PCR.The developed quantitative RT-PCR assay could detect 5.0×10~(0) copy/μL of plasmid DNA and its sensitivity was 100 times higher than that of the routine RT-PCR,while the results of the quantitative RT-PCR were the same as that of the routine RT-PCR.Three samples were examined using the real-time RT-PCR repeatedly and the results indicated that the real-time quantitative RT-PCR was reproducible and could be used for the(diagnosis) of PRRSV infection.
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