爱华网在血液检测的必检项目中有三个是跟病毒有关的，HIV-1，HBV和HCV。直接检测血样中病毒的核酸，可以缩短窗口期，降低漏检率。
目前使用较多的是核酸检测技术（nucleic acid amplification testing,NAT）方法包括两种扩增方式：对靶核酸直接扩增，即PCR法和和转录介导的扩增方法。
转录介导的扩增方法(transcription mediated amplification,TMA)首先是由Dr.LarryMimms
[Sensitive detection of genetic variants of HIV-1 andHCV with an HIV-1/HCV assay based on transcription-mediatedamplification. J Virol Methods. 2002 ：102(1-2):139-55]发明的，其公司Gen-Probe2004年因此获得美国国家技术奖（NationalMedal of Technology）。
TMA是一种利用Money鼠白血病病毒（MMLV）逆转录酶和T7 RNA多聚酶2种酶的共同作用,在等温条件下来扩增RNA或DNA的反应系统,主要扩增原理为:目标序列在逆转录酶作用下,以引物为引导进行逆转录, 逆转录酶的RNA酶H活性将杂合链上的RNA降解以后,合成双链的DNA,并在T7RNA多聚酶作用下,转录出成千上万个目标RNA序列,这些RNA又可以作为模板进行下一个循环,整个反应是一个自催化过程。NASBA的原理与TMA相似,只是在核酸提取和扩增产物检测的方法上有所不同。这两种方法的特异性强,灵敏度高,反应条件简单,扩增效率高,无需专门的扩增仪器,且因整个反应在1个试管中进行,也减少了污染。
google搜索时从发明人本科毕业的学校Davidson College(全美十大文理学院之一）网站上TMA的流程图。
一扩增
Step1. Mix allingredients.
The biopsy cells are lysed, and the amplification begins whenprimer#1 binds to the target template (HIV's RNA genome). RTsynthesizes a complementary DNA strand and degrades the HIV genometemplate
Step2. ThePrimer#1 binds, and RT binds to 3'end ofprimer.
Note that primer #1 has a small extension that does not bind toHIV
Step3. RTsynthesizes first strand of cDNA (left) and degrades RNA template(right).
Notethat these two steps are not separated in reality but are done sohere for illustration purposes only.
Once thefirst cDNA strand is synthesized, a second primer binds, and RTproduces double stranded cDNA that also includes the smallextension from primer #1. This extension is a promoter recognizedby the RNA polymerase included in the TMA reaction.
Step4. Primer#2 binds to the cDNA, and RT begins synthesis for the second strandof cDNA.
Step5. RT synthesizes thesecond strand of cDNA including the promoter extension of primer#1.
Now the RNApolymerase is able to bind to the dsDNA promoter sequence and begintranscription. Transcription happens many times to amplify theoriginal one HIV template by 100 to 1000 fold.
Step6. RNApolymerase binds to the promoter sequence of thedsDNA.
Step7. RNA polymeraseproduces a new copy of the original template (HIVgenome).
Step8.Transcription happens 100 - 1000 times to amplify the originaltemplate.
In the end, each of the newlysynthesized RNA templates serve as starting material for step 2above, and the entire process is repeated over and over again.
Note that unlike PCR, the reactionremains at a constanttemperature! Within 15 - 30 minutes over 10billion copies of the HIV RNA are produced in a single tube.
二、检测
Once the RNA is amplified, it needsto be detected. To do this, the amplicons are incubated with aprobe of DNA that is covalently modified with acridinium ester(AE). Any probe that is not hybridized with the RNA will lose itsAE when exposed to hydroxide ions (high pH).
Step9. Thereaction is stopped and mixed with a ssDNA probe(left) thatcontains adridinium easter ( AE; right)marker.(吖啶酯是一类可用作化学发光标记物的化学物质。)
Step10.Some probe willhybridize3 to the amplified RNA and protect the AE fromdestruction. Unprotected AE is cleaved from the probe anddegraded.
If theprobe hybridized with an RNA amplicon, then the AE is protected andwill produce light in the presence of a superoxide. This light canbe measured and is proportional to the number of template molecules(HIV genomes) in the initialreaction.
Step11. When mixed with the appropriatesubstrate, the AE is modified and emits light as a product of thechemical reaction.
