兩種標(biāo)簽引物RT-PCR結(jié)合Sanger測(cè)序檢測(cè)登革病毒的研究
發(fā)布時(shí)間：2018-08-24 19:20
【摘要】： 建立兩種標(biāo)簽引物RT-PCR結(jié)合Sanger測(cè)序的方法,檢測(cè)登革病毒,包括新變異的登革病毒。一種方法可以快速診斷急性期單一血清型感染的登革熱,另一種方法可以診斷雙重血清型感染的登革熱。 3’末端錨定-標(biāo)簽引物RT-PCR(NAT-PCR)結(jié)合Sanger測(cè)序法是利用登革病毒(“正鏈”)3’最末端的保守序列作為錨定靶點(diǎn)進(jìn)行反轉(zhuǎn)錄,合成帶有3’最末端序列的cDNA(“負(fù)鏈”)(RT步驟),以登革病毒特異性的簡(jiǎn)并引物合成cDNA的第二條鏈(SS步驟)。由于cDNA合成引物與簡(jiǎn)并引物的5’末端均帶有人工設(shè)計(jì)的“標(biāo)簽”序列,所以新合成的第二條鏈(“正鏈”)的兩端分別帶有彼此互補(bǔ)的“標(biāo)簽”序列,再以“標(biāo)簽”序列作為引物進(jìn)行PCR擴(kuò)增(AMP步驟)。擴(kuò)增產(chǎn)物帶有的相同3’最末端序列(cDNA合成引物)作為測(cè)序引物進(jìn)行Sanger測(cè)序。并利用SYBR Green I real-time PCR進(jìn)行監(jiān)測(cè)和篩選NAT-PCR產(chǎn)物及其反應(yīng)參數(shù),優(yōu)化出最佳的NAT-PCR擴(kuò)增效率以及最快速的Sanger測(cè)序程序。該方法對(duì)A型流感病毒、拉沙病毒、西尼羅病毒、乙型腦炎病毒、腎綜合征出血熱病毒和黃熱病病毒檢測(cè)不到可讀序列,具有較好的特異性;對(duì)四個(gè)血清型登革病毒RNA的檢出限為11-31個(gè)拷貝/反應(yīng)(兩步NAT-PCR)或110-310個(gè)拷貝/反應(yīng)(一步NAT-PCR),敏感性較高。對(duì)25份臨床血清樣本RNA的檢測(cè)結(jié)果與臨床診斷一致,可獲得400-520bp的可讀序列,其中包括3份登革I型、5份登革II型、3份登革III型病毒陽(yáng)性樣本,其他樣本均為陰性結(jié)果。一步NAT-PCR比兩步NAT-PCR獲得檢測(cè)結(jié)果好:陽(yáng)性檢出率較高、可讀序列更長(zhǎng)。 隨機(jī)PCR方法也是利用標(biāo)簽引物進(jìn)行RT-PCR,擴(kuò)增的步驟與NAT-PCR方法相同,但以隨機(jī)引物進(jìn)行反轉(zhuǎn)錄和cDNA第二條鏈的合成。獲得的擴(kuò)增產(chǎn)物進(jìn)行TA克隆,以單克隆的M13 PCR產(chǎn)物為模板進(jìn)行Sanger測(cè)序。通過優(yōu)化反應(yīng)條件,并利用SYBR Green I real-time PCR和Bioanalyzer DNA質(zhì)量分析儀進(jìn)行監(jiān)測(cè)和篩選,優(yōu)化出無偏嗜擴(kuò)增的隨機(jī)PCR反應(yīng)模式,可對(duì)雙重感染以及因RNA降解而缺失3’末端序列的所有登革病毒進(jìn)行檢測(cè)。該方法不僅可以檢測(cè)登革病毒,還可以檢測(cè)樣本中其他的單鏈不分節(jié)段RNA病毒以及該類型的未知病毒。其測(cè)序鑒定登革病毒的敏感性為100個(gè)拷貝的RNA/μl血清。檢測(cè)DENV-1和DENV-2的混合感染樣本,Sanger測(cè)序鑒定的陽(yáng)性克隆率分別為21/92和32/96。對(duì)25份臨床血清樣本RNA的陽(yáng)性檢測(cè)結(jié)果與臨床診斷一致,也與NAT-PCR結(jié)合Sanger測(cè)序的檢測(cè)結(jié)果一致;但在陰性樣本中,有1例被鑒定含有丙型肝炎病毒。在所有的陽(yáng)性樣本中,沒有發(fā)現(xiàn)雙重血清型登革病毒感染的樣本。 NAT-PCR結(jié)合Sanger測(cè)序方法,通過錨定3’末端高度保守的序列,以單管反應(yīng)同時(shí)鑒定四個(gè)血清型的登革病毒及其新變異毒株。該方法可在5個(gè)小時(shí)內(nèi)完成從樣本RNA的提取到序列分析的全過程,檢測(cè)特異性強(qiáng)、敏感性高,具有廣泛的臨床應(yīng)用價(jià)值。由于在登革熱流行非常嚴(yán)重的地方會(huì)出現(xiàn)雙重感染的情況,為此本研究建立了隨機(jī)PCR結(jié)合TA克隆和Sanger測(cè)序的方法可診斷雙重血清型登革病毒的混合感染,并具有檢測(cè)新的病毒變異株以及未知病毒的能力,有助于發(fā)現(xiàn)由單股正鏈不分節(jié)段的RNA病毒引起的人或動(dòng)物傳染病的混合感染。
[Abstract]:To establish two methods of RT-PCR and Sanger sequencing with labeled primers for detection of dengue virus, including newly mutated dengue virus. One method can rapidly diagnose acute single serotype infection of dengue fever, the other can diagnose double serotype infection of dengue fever.
3'-terminal anchoring-tagging primer RT-PCR (NAT-PCR) combined with Sanger sequencing is a method of reverse transcription using the conserved sequence of the 3'-terminal end of dengue virus ("positive chain") as anchoring target to synthesize a 3'-terminal sequence of cDNA ("negative chain") (RT step), and to synthesize a second DNA strand (SS step) with a dengue virus-specific degenerate primer. Because the 5'end of the synthetic primer and degenerate primer of the cDNA contain the designed "tag" sequence, the two ends of the newly synthesized second strand ("positive strand") have complementary "tag" sequence respectively, and then the "tag" sequence is used as the primer for PCR amplification (AMP step). The same 3'end sequence of the amplified product is carried out. Sequencing of influenza A virus, Lassa virus, West Nile virus and encephalitis B virus was carried out by using SYBR Green I real-time PCR to monitor and screen NAT-PCR products and their reaction parameters. Hemorrhagic fever with renal syndrome virus and yellow fever virus can not detect the readable sequence, with good specificity; the detection limit of four serotypes of dengue virus RNA is 11-31 copies / reactions (two-step NAT-PCR) or 110-310 copies / reactions (one-step NAT-PCR), with high sensitivity. The detection results of RNA in 25 clinical serum samples and clinical diagnosis 1. As a result, 400-520 BP readable sequences were obtained, including 3 dengue type I, 5 dengue type II, 3 dengue type III virus positive samples, and all the other samples were negative.
Random PCR was also used for RT-PCR with labeled primers. The amplification procedure was the same as that of NAT-PCR, but the reverse transcription and the synthesis of the second strand of the cDNA were carried out with random primers. The amplified product was cloned by TA and sequenced by Sanger using the template of the monoclonal M13 PCR product. The e-PCR and Bioanalyzer DNA quality analyzer were used to monitor and screen the dengue virus and optimize the random PCR reaction mode of unbiased amplification. The method can detect all dengue viruses with double infection and deletion of 3'terminal sequence due to RNA degradation. The positive cloning rates of DENV-1 and DENV-2 were 21/92 and 32/96 respectively. The positive detection results of RNA in 25 clinical serum samples were consistent with clinical diagnosis and NAT-PCR. The results were consistent with Sanger sequencing, but one of the negative samples was identified as having hepatitis C virus. No double serotype dengue virus infection was found in all positive samples.
Four serotypes of dengue virus and its new variant strains were simultaneously identified by single-tube reaction by anchoring highly conserved 3'-terminal sequences with NAT-PCR combined with Sanger sequencing method. The method can complete the whole process from RNA extraction to sequence analysis within 5 hours. The method has strong specificity and high sensitivity, and has wide clinical application. Value. Because double infection occurs in places where dengue fever is very serious, a random PCR combined with TA cloning and Sarger sequencing method was established to diagnose mixed infection of double serotype dengue virus. It has the ability to detect new virus variants and unknown viruses, and is helpful to detect positive single strand dengue virus. A mixed infection of human or animal infectious diseases caused by RNA virus.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R373
本文編號(hào)：2201783
[Abstract]:To establish two methods of RT-PCR and Sanger sequencing with labeled primers for detection of dengue virus, including newly mutated dengue virus. One method can rapidly diagnose acute single serotype infection of dengue fever, the other can diagnose double serotype infection of dengue fever.
3'-terminal anchoring-tagging primer RT-PCR (NAT-PCR) combined with Sanger sequencing is a method of reverse transcription using the conserved sequence of the 3'-terminal end of dengue virus ("positive chain") as anchoring target to synthesize a 3'-terminal sequence of cDNA ("negative chain") (RT step), and to synthesize a second DNA strand (SS step) with a dengue virus-specific degenerate primer. Because the 5'end of the synthetic primer and degenerate primer of the cDNA contain the designed "tag" sequence, the two ends of the newly synthesized second strand ("positive strand") have complementary "tag" sequence respectively, and then the "tag" sequence is used as the primer for PCR amplification (AMP step). The same 3'end sequence of the amplified product is carried out. Sequencing of influenza A virus, Lassa virus, West Nile virus and encephalitis B virus was carried out by using SYBR Green I real-time PCR to monitor and screen NAT-PCR products and their reaction parameters. Hemorrhagic fever with renal syndrome virus and yellow fever virus can not detect the readable sequence, with good specificity; the detection limit of four serotypes of dengue virus RNA is 11-31 copies / reactions (two-step NAT-PCR) or 110-310 copies / reactions (one-step NAT-PCR), with high sensitivity. The detection results of RNA in 25 clinical serum samples and clinical diagnosis 1. As a result, 400-520 BP readable sequences were obtained, including 3 dengue type I, 5 dengue type II, 3 dengue type III virus positive samples, and all the other samples were negative.
Random PCR was also used for RT-PCR with labeled primers. The amplification procedure was the same as that of NAT-PCR, but the reverse transcription and the synthesis of the second strand of the cDNA were carried out with random primers. The amplified product was cloned by TA and sequenced by Sanger using the template of the monoclonal M13 PCR product. The e-PCR and Bioanalyzer DNA quality analyzer were used to monitor and screen the dengue virus and optimize the random PCR reaction mode of unbiased amplification. The method can detect all dengue viruses with double infection and deletion of 3'terminal sequence due to RNA degradation. The positive cloning rates of DENV-1 and DENV-2 were 21/92 and 32/96 respectively. The positive detection results of RNA in 25 clinical serum samples were consistent with clinical diagnosis and NAT-PCR. The results were consistent with Sanger sequencing, but one of the negative samples was identified as having hepatitis C virus. No double serotype dengue virus infection was found in all positive samples.
Four serotypes of dengue virus and its new variant strains were simultaneously identified by single-tube reaction by anchoring highly conserved 3'-terminal sequences with NAT-PCR combined with Sanger sequencing method. The method can complete the whole process from RNA extraction to sequence analysis within 5 hours. The method has strong specificity and high sensitivity, and has wide clinical application. Value. Because double infection occurs in places where dengue fever is very serious, a random PCR combined with TA cloning and Sarger sequencing method was established to diagnose mixed infection of double serotype dengue virus. It has the ability to detect new virus variants and unknown viruses, and is helpful to detect positive single strand dengue virus. A mixed infection of human or animal infectious diseases caused by RNA virus.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R373
【相似文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 胡哲;兩種標(biāo)簽引物RT-PCR結(jié)合Sanger測(cè)序檢測(cè)登革病毒的研究[D];吉林大學(xué);2009年
，本文編號(hào)：2201783
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