藍(lán)舌病病毒生物條形碼及熒光定量型生物條形碼檢測(cè)體系的建立與評(píng)價(jià)
發(fā)布時(shí)間：2018-08-17 11:20
【摘要】： 藍(lán)舌病(bluetongue, BT)是由藍(lán)舌病病毒(blue tongue virus, BTV)引起的一種侵害反芻動(dòng)物的蟲(chóng)媒性傳染病,被世界動(dòng)物衛(wèi)生組織(World Organization for Animal Health, OIE)列為上報(bào)疾病,我國(guó)將其列為一類動(dòng)物疫病,是國(guó)內(nèi)外動(dòng)物防疫和出入境檢驗(yàn)檢疫機(jī)構(gòu)監(jiān)測(cè)和防控的重點(diǎn)疫病。此外,藍(lán)舌病病毒還是人用動(dòng)物源生物制品(如人用牛源凝血酶、凝血因子Ⅷ、聚合牛血紅蛋白、胸腺肽)和生物制品敷料(如牛源明膠、牛源膠原)病毒安全性檢測(cè)的病毒之一。 BTV屬于呼腸孤病毒科(Reoviridae)環(huán)狀病毒屬(Orbivirus)成員,至少存在24個(gè)血清型,為血清型多樣性,呈全球性分布。隨著全球氣溫變暖,2006年～2008年許多國(guó)家爆發(fā)藍(lán)舌病,使其分布范圍不斷擴(kuò)張,并出現(xiàn)了地域性新血清型,而現(xiàn)在的檢測(cè)試劑存在著的靈敏度不高覆蓋血清型范圍窄的缺點(diǎn)阻礙了BTV的病原監(jiān)控工作。 因此,建立高靈敏度的適于各血清型BTV的特異性檢測(cè)技術(shù),以及研制相應(yīng)的檢測(cè)試劑盒已經(jīng)迫在眉睫。 生物條形碼檢測(cè)技術(shù)(bio-bar codes assay, BCA)是2003年美國(guó)科學(xué)家Mirkin領(lǐng)導(dǎo)的課題組首次報(bào)道的一種新型標(biāo)記免疫測(cè)定技術(shù),其突出特點(diǎn)是具有極高的靈敏度,其基本原理幾乎等同于ELISA檢測(cè)中雙抗體夾心法高度特異地捕獲抗原,通過(guò)檢測(cè)特異條形碼DNA鏈(bar code DNA)而實(shí)現(xiàn)對(duì)抗原物質(zhì)的間接檢測(cè)。對(duì)特異條形碼DNA鏈的檢測(cè)采用常規(guī)PCR擴(kuò)增或基于金標(biāo)銀染的芯片檢測(cè),由于PCR擴(kuò)增和芯片檢測(cè)的放大效應(yīng),使得檢測(cè)靈敏度得到極大提高,可達(dá)常規(guī)ELISA方法的106倍。 熒光定量PCR技術(shù)(fluorescent quantitative PCR, FQ-PCR)是通過(guò)在PCR反應(yīng)體系中加入熒光基團(tuán),利用熒光信號(hào)的變化實(shí)時(shí)檢測(cè)PCR擴(kuò)增反應(yīng)中每一個(gè)循環(huán)擴(kuò)增產(chǎn)物量的變化,通過(guò)Ct值和標(biāo)準(zhǔn)曲線的分析對(duì)起始模板進(jìn)行定量分析,被認(rèn)為是當(dāng)今核酸定量的金標(biāo)準(zhǔn)。 本研究針對(duì)藍(lán)舌病毒的檢測(cè)現(xiàn)狀,建立了BTV的BCA檢測(cè)體系和BTV的熒光定量型生物條形碼檢測(cè)體系(fluorescent quantitative bio-bar codes assay, FQ-BCA),并對(duì)檢測(cè)體系進(jìn)行了靈敏度、特異性、重復(fù)性等方法學(xué)評(píng)價(jià)。 本研究主要包括以下幾部分: 1. NP探針制備、純化及鑒定 利用檸檬酸三鈉還原法制備金納米顆粒,并對(duì)金納米顆粒進(jìn)行光學(xué)、TEM、UV-vis和濃度鑒定;利用BTV全病毒免疫新西蘭大白兔制備BTV多抗,純化后進(jìn)行濃度、SDS-PAGE電泳和效價(jià)鑒定;利用金納米顆粒、BTV多抗和人工合成的互補(bǔ)probe NP鏈、條形碼DNA鏈制備NP探針,純化后進(jìn)行TEM、UV-vis和濃度鑒定。 2. MMP探針制備、純化及鑒定 利用BTV雜交瘤細(xì)胞株免疫BALB/c小鼠制備BTV單抗,純化后進(jìn)行濃度、效價(jià)和SDS-PAGE鑒定;利用制備的BTV單抗和磁性微球制備MMP探針,并對(duì)探針進(jìn)行標(biāo)記效率鑒定。 3.檢測(cè)探針制備、純化及鑒定 利用金納米顆粒和人工合成的NP標(biāo)簽鏈制備檢測(cè)探針,純化后對(duì)檢測(cè)探針進(jìn)行TEM、UV-vis和濃度鑒定。 4. BTV的BCA及FQ-BCA檢測(cè)體系的建立及優(yōu)化 利用NP探針和MMP探針通過(guò)抗原抗體作用制備NP-VP7-MMP三明治復(fù)合物,在高溫低鹽條件下,釋放條形碼DNA鏈。對(duì)獲得的條形碼DNA鏈分別進(jìn)行常規(guī)PCR檢測(cè)、芯片檢測(cè)和FQ-PCR檢測(cè),并對(duì)檢測(cè)條件進(jìn)行優(yōu)化。 5. BCA及FQ-BCA檢測(cè)體系的靈敏度評(píng)價(jià) VP7蛋白檢測(cè)靈敏度評(píng)價(jià):ELISA方法檢測(cè)靈敏度為1ng/mL;BCA檢測(cè)體系的常規(guī)PCR檢測(cè)靈敏度為1fg/mL,芯片檢測(cè)靈敏度為10fg/mL;FQ-BCA檢測(cè)體系的檢測(cè)靈敏度為100ag/mL,靈敏度分別為常規(guī)ELISA方法的106、105、10~7倍。 BTV全病毒檢測(cè)靈敏度評(píng)價(jià):ELISA方法檢測(cè)靈敏度為102TCID50;BCA檢測(cè)體系的常規(guī)PCR檢測(cè)靈敏度為10-4TCID50,芯片檢測(cè)靈敏度為10-3TCID50;FQ-BCA檢測(cè)體系的檢測(cè)靈敏度為10-5TCID50,靈敏度分別為常規(guī)ELISA方法的106、105、107倍。 結(jié)果表明BCA和FQ-BCA檢測(cè)體系的檢測(cè)靈敏度遠(yuǎn)遠(yuǎn)超過(guò)現(xiàn)在的蛋白檢測(cè)首選方法——ELISA方法。 6. BCA及FQ-BCA檢測(cè)體系的特異性評(píng)價(jià) 用建立的BCA和FQ-BCA檢測(cè)體系對(duì)BTV、EHDV、BVDV進(jìn)行特異性交叉試驗(yàn)。結(jié)果為:BCA及FQ-BCA檢測(cè)體系均具有良好的特異性。 7. BCA及FQ-BCA檢測(cè)體系的重復(fù)性評(píng)價(jià) BTV滴度為10-1TCID50時(shí):BCA檢測(cè)體系的芯片檢測(cè)的批內(nèi)CV值分別為4.2%、4.1%、3.4%,批間CV值為6.3%;FQ-BCA檢測(cè)體系的批內(nèi)CV值分別為2.06%、1.45%、1.43%,批間CV值為2.52%。 BTV滴度為10-2TCID50時(shí):BCA檢測(cè)體系芯片檢測(cè)的批內(nèi)CV值分別為4.2%、3.9%、4.4%,批間CV值為5.5%;FQ-BCA檢測(cè)體系的批內(nèi)CV值分別為1.95%、1.37%、1.56%,批間CV值為2.1%。 BTV滴度為10-3TCID50時(shí):BCA檢測(cè)體系的芯片檢測(cè)的批內(nèi)CV值分別為4.1%、4.4%、3.8%,批間CV值為6.2%;FQ-BCA檢測(cè)體系的批內(nèi)CV值分別為1.43%、1.30%、1.19%,批間CV值為1.91%。 結(jié)果表明BCA及FQ-BCA檢測(cè)體系都具有良好的批內(nèi)和批間重復(fù)性,且FQ-BCA檢測(cè)體系重復(fù)性更好。 綜上所述,本研究成功地建立了BTV的BCA檢測(cè)體系和BTV的FQ-BCA檢測(cè)體系,并對(duì)體系進(jìn)行了方法學(xué)評(píng)價(jià)。其意義在于:1)本研究為研制BTV的BCA檢測(cè)試劑和FQ-BCA檢測(cè)試劑奠定了基礎(chǔ);2)本研究為其它類似的被檢物尤其是那些不適于建立核酸檢測(cè)體系的微量物質(zhì)如毒素、生物戰(zhàn)劑、類固醇、脂類、維生素、腫瘤特異性標(biāo)志物等的檢測(cè)提供了新方法,對(duì)被檢物建立類似檢測(cè)體系起到良好的示范作用。
[Abstract]:Bluetongue disease (BT) is an insect-borne infectious disease caused by blue tongue virus (BTV) which infects ruminants. It is listed as a reported disease by the World Organization for Animal Health (OIE). It is classified as a kind of animal epidemic disease in China and abroad, and it is also an animal quarantine and entry-exit inspection and quarantine. Bluetongue virus is also one of the viruses used to detect the safety of human animal-derived biological products (such as human bovine thrombin, coagulation factor_, polymerized bovine hemoglobin, thymosin) and biological dressings (such as bovine gelatin, bovine collagen).
BTV belongs to the Orbivirus family of Reoviridae. There are at least 24 serotypes of BTV. BTV is a globally distributed serotype with diverse serotypes. The presence of low sensitivity and narrow coverage of serotypes limits the monitoring of BTV pathogens.
Therefore, it is imminent to establish a highly sensitive and specific detection technique for BTV of various serotypes and develop corresponding detection kits.
Bio-bar codes assay (BCA) is a new labeled immunoassay technique first reported by a research group led by American scientist Mirkin in 2003. Its outstanding feature is its high sensitivity. Its basic principle is almost equal to the double antibody sandwich method in ELISA detection, which can capture antigens with high specificity. Specific bar code DNA strands (bar code DNA) can be used for indirect detection of antigen substances. Detection of specific bar code DNA strands using conventional PCR amplification or gold labeled silver staining chip detection, due to the amplification effect of PCR amplification and chip detection, the detection sensitivity has been greatly improved, up to 106 times the conventional ELISA method.
Fluorescent quantitative polymerase chain reaction (FQ-PCR) is a real-time detection of changes in the amount of each amplified product in a PCR amplification reaction by adding fluorescent groups to the PCR reaction system. The initial template is quantitatively analyzed by the analysis of CT value and standard curve. FQ-PCR is considered to be today's nucleic acid. Quantitative gold standard.
In this study, a BCA detection system for BTV and a fluorescent quantitative bio-bar codes assay (FQ-BCA) for BTV were established, and the sensitivity, specificity and repeatability of the detection system were evaluated.
This study mainly includes the following parts:
Preparation, purification and identification of 1. NP probe
Gold nanoparticles were prepared by trisodium citrate reduction method, and the gold nanoparticles were identified by optical, TEM, UV-vis and concentration analysis; BTV polyclonal antibodies were prepared by immunizing New Zealand white rabbits with BTV whole virus, then purified, SDS-PAGE electrophoresis and titer identification; gold nanoparticles, BTV polyclonal antibodies and synthetic complementary NP chains, bar code D were used. NA chain was used to prepare NP probe. After purification, TEM, UV-vis and concentration were identified.
Preparation, purification and identification of 2. MMP probe
BTV monoclonal antibody was prepared by immunizing BALB/c mice with BTV hybridoma cell line, then purified and identified by concentration, titer and SDS-PAGE. MMP probe was prepared by using BTV monoclonal antibody and magnetic microspheres, and the labeling efficiency of the probe was evaluated.
3. detection, preparation, purification and identification of probes
Gold nanoparticles and synthetic NP tag chains were used to prepare detection probes. After purification, the probe was identified by TEM, UV-vis and concentration.
Establishment and optimization of BCA and FQ-BCA detection system for 4. BTV
NP-VP7-MMP sandwich complex was prepared by antigen-antibody interaction of NP probe and MMP probe, and barcode DNA strands were released under high temperature and low salt conditions. The obtained barcode DNA strands were detected by conventional PCR, chip detection and FQ-PCR respectively, and the detection conditions were optimized.
Sensitivity evaluation of 5. BCA and FQ-BCA detection system
VP7 protein detection sensitivity evaluation: ELISA detection sensitivity is 1ng/mL; BCA detection system of conventional PCR detection sensitivity is 1fg/mL, chip detection sensitivity is 10fg/mL; FQ-BCA detection system detection sensitivity is 100ag/mL, sensitivity is 106,105,10-7 times that of conventional ELISA method.
BTV whole virus detection sensitivity evaluation: ELISA detection sensitivity of 102 TCID 50; BCA detection system of conventional PCR detection sensitivity of 10-4 TCID 50, chip detection sensitivity of 10-3 TCID 50; FQ-BCA detection system of 10-5 TCID 50, sensitivity of 106, 105, 107 times as conventional ELISA method.
The results showed that the sensitivity of BCA and FQ-BCA detection system was much higher than that of ELISA method, which is the preferred method for protein detection.
Specificity evaluation of 6. BCA and FQ-BCA detection system
BCA and FQ-BCA were used to test BTV, EHDV and BVDV. The results showed that BCA and FQ-BCA had good specificity.
Repeatability evaluation of 7. BCA and FQ-BCA detection system
When BTV titer was 10-1 TCID50, the CV values of BCA chip were 4.2%, 4.1%, 3.4% and 6.3% respectively, while that of FQ-BCA chip was 2.06%, 1.45%, 1.43% and 2.52% respectively.
When BTV titer was 10-2TCID50, the CV values of BCA chip were 4.2%, 3.9%, 4.4% and 5.5% respectively, while that of FQ-BCA chip was 1.95%, 1.37%, 1.56% and 2.1% respectively.
When BTV titer was 10-3 TCID50, the intra-batch CV was 4.1%, 4.4%, 3.8% and the inter-batch CV was 6.2% for BCA, 1.43%, 1.30%, 1.19% and 1.91% for FQ-BCA, respectively.
The results showed that both BCA and FQ-BCA detection systems had good intra-batch and inter-batch repeatability, and FQ-BCA detection system had better repeatability.
To sum up, the BCA detection system for BTV and the FQ-BCA detection system for BTV were successfully established, and the methodological evaluation of the system was carried out. The detection system of trace substances such as toxins, biological warfare agents, steroids, lipids, vitamins, tumor specific markers and so on provides a new method for the detection of the detection system, which plays a good role in establishing a similar detection system.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R373
本文編號(hào)：2187458
[Abstract]:Bluetongue disease (BT) is an insect-borne infectious disease caused by blue tongue virus (BTV) which infects ruminants. It is listed as a reported disease by the World Organization for Animal Health (OIE). It is classified as a kind of animal epidemic disease in China and abroad, and it is also an animal quarantine and entry-exit inspection and quarantine. Bluetongue virus is also one of the viruses used to detect the safety of human animal-derived biological products (such as human bovine thrombin, coagulation factor_, polymerized bovine hemoglobin, thymosin) and biological dressings (such as bovine gelatin, bovine collagen).
BTV belongs to the Orbivirus family of Reoviridae. There are at least 24 serotypes of BTV. BTV is a globally distributed serotype with diverse serotypes. The presence of low sensitivity and narrow coverage of serotypes limits the monitoring of BTV pathogens.
Therefore, it is imminent to establish a highly sensitive and specific detection technique for BTV of various serotypes and develop corresponding detection kits.
Bio-bar codes assay (BCA) is a new labeled immunoassay technique first reported by a research group led by American scientist Mirkin in 2003. Its outstanding feature is its high sensitivity. Its basic principle is almost equal to the double antibody sandwich method in ELISA detection, which can capture antigens with high specificity. Specific bar code DNA strands (bar code DNA) can be used for indirect detection of antigen substances. Detection of specific bar code DNA strands using conventional PCR amplification or gold labeled silver staining chip detection, due to the amplification effect of PCR amplification and chip detection, the detection sensitivity has been greatly improved, up to 106 times the conventional ELISA method.
Fluorescent quantitative polymerase chain reaction (FQ-PCR) is a real-time detection of changes in the amount of each amplified product in a PCR amplification reaction by adding fluorescent groups to the PCR reaction system. The initial template is quantitatively analyzed by the analysis of CT value and standard curve. FQ-PCR is considered to be today's nucleic acid. Quantitative gold standard.
In this study, a BCA detection system for BTV and a fluorescent quantitative bio-bar codes assay (FQ-BCA) for BTV were established, and the sensitivity, specificity and repeatability of the detection system were evaluated.
This study mainly includes the following parts:
Preparation, purification and identification of 1. NP probe
Gold nanoparticles were prepared by trisodium citrate reduction method, and the gold nanoparticles were identified by optical, TEM, UV-vis and concentration analysis; BTV polyclonal antibodies were prepared by immunizing New Zealand white rabbits with BTV whole virus, then purified, SDS-PAGE electrophoresis and titer identification; gold nanoparticles, BTV polyclonal antibodies and synthetic complementary NP chains, bar code D were used. NA chain was used to prepare NP probe. After purification, TEM, UV-vis and concentration were identified.
Preparation, purification and identification of 2. MMP probe
BTV monoclonal antibody was prepared by immunizing BALB/c mice with BTV hybridoma cell line, then purified and identified by concentration, titer and SDS-PAGE. MMP probe was prepared by using BTV monoclonal antibody and magnetic microspheres, and the labeling efficiency of the probe was evaluated.
3. detection, preparation, purification and identification of probes
Gold nanoparticles and synthetic NP tag chains were used to prepare detection probes. After purification, the probe was identified by TEM, UV-vis and concentration.
Establishment and optimization of BCA and FQ-BCA detection system for 4. BTV
NP-VP7-MMP sandwich complex was prepared by antigen-antibody interaction of NP probe and MMP probe, and barcode DNA strands were released under high temperature and low salt conditions. The obtained barcode DNA strands were detected by conventional PCR, chip detection and FQ-PCR respectively, and the detection conditions were optimized.
Sensitivity evaluation of 5. BCA and FQ-BCA detection system
VP7 protein detection sensitivity evaluation: ELISA detection sensitivity is 1ng/mL; BCA detection system of conventional PCR detection sensitivity is 1fg/mL, chip detection sensitivity is 10fg/mL; FQ-BCA detection system detection sensitivity is 100ag/mL, sensitivity is 106,105,10-7 times that of conventional ELISA method.
BTV whole virus detection sensitivity evaluation: ELISA detection sensitivity of 102 TCID 50; BCA detection system of conventional PCR detection sensitivity of 10-4 TCID 50, chip detection sensitivity of 10-3 TCID 50; FQ-BCA detection system of 10-5 TCID 50, sensitivity of 106, 105, 107 times as conventional ELISA method.
The results showed that the sensitivity of BCA and FQ-BCA detection system was much higher than that of ELISA method, which is the preferred method for protein detection.
Specificity evaluation of 6. BCA and FQ-BCA detection system
BCA and FQ-BCA were used to test BTV, EHDV and BVDV. The results showed that BCA and FQ-BCA had good specificity.
Repeatability evaluation of 7. BCA and FQ-BCA detection system
When BTV titer was 10-1 TCID50, the CV values of BCA chip were 4.2%, 4.1%, 3.4% and 6.3% respectively, while that of FQ-BCA chip was 2.06%, 1.45%, 1.43% and 2.52% respectively.
When BTV titer was 10-2TCID50, the CV values of BCA chip were 4.2%, 3.9%, 4.4% and 5.5% respectively, while that of FQ-BCA chip was 1.95%, 1.37%, 1.56% and 2.1% respectively.
When BTV titer was 10-3 TCID50, the intra-batch CV was 4.1%, 4.4%, 3.8% and the inter-batch CV was 6.2% for BCA, 1.43%, 1.30%, 1.19% and 1.91% for FQ-BCA, respectively.
The results showed that both BCA and FQ-BCA detection systems had good intra-batch and inter-batch repeatability, and FQ-BCA detection system had better repeatability.
To sum up, the BCA detection system for BTV and the FQ-BCA detection system for BTV were successfully established, and the methodological evaluation of the system was carried out. The detection system of trace substances such as toxins, biological warfare agents, steroids, lipids, vitamins, tumor specific markers and so on provides a new method for the detection of the detection system, which plays a good role in establishing a similar detection system.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R373
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