36:
non-isothermal and isothermal. Non-isothermal amplification produces multiple copies of RNA/DNA through reiterative cycling between different temperatures. Isothermal amplification produces multiple copies of RNA/DNA at a constant reaction temperature. NASBA takes single stranded RNA, anneals primers to it at 65°C, and then amplifies it at 41°C to produce multiple copies of single stranded RNA. In order for successful amplification to occur, an enzyme cocktail containing, Avian
Myeloblastosis Reverse Transcriptase (AMV-RT), RNase H, and RNA polymerase is used. AMV-RT synthesizes a complementary DNA strand (cDNA) from the RNA template once the primer is annealed. RNase H then degrades the RNA template and the other primer binds to the cDNA to form double stranded DNA, which RNA polymerase uses to synthesize copies of RNA. One key aspect of NASBA is that the starting material and end product is always single stranded RNA. That being said, it can be used to amplify DNA, but the DNA must be translated into RNA in order for successful amplification to occur.
59:(in order to synthesize a complementary DNA strand as a template), NASBA's main advantage is that it works under isothermal conditions – usually at a constant temperature of 41 °C or two different temperatures, depending on the primers and enzymes used. Even when two different temperatures are applied, it is still considered isothermal, because it does not cycle back and forth between those temperatures. NASBA can be used in medical diagnostics as an alternative to PCR that is quicker and more sensitive in some circumstances.
100:
binds to the promoter region on the double strand. Since T7 RNA polymerase can only transcribe in the 3' to 5' direction the sense DNA is transcribed and an anti-sense RNA is produced. This is repeated, and the polymerase continuously produces complementary RNA strands of this template which results
50:
NASBA was developed by J Compton in 1991, who defined it as "a primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature". Immediately after the invention of NASBA it was used for the rapid diagnosis and quantification of
35:
Nucleic acid amplification is a technique used to produce several copies of a specific segment of RNA/DNA. Amplified RNA and DNA can be used for a variety of applications, such as genotyping, sequencing, and detection of bacteria or viruses. There are two different types of amplification,
706:
Kievits, T; Van Gemen, B; Van Strijp, D; Schukkink, R; Dircks, M; Adriaanse, H; Malek, L; Sooknanan, R; Lens, P (1991). "NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection".
27:
which is used to produce multiple copies of single stranded RNA. NASBA is a two-step process that takes RNA and anneals specially designed primers, then utilizes an enzyme cocktail to amplify it.
971:
Böhmer, A; Schildgen, V; Lüsebrink, J; Ziegler, S; Tillmann, RL; Kleines, M; Schildgen, O (2009). "Novel application for isothermal nucleic acid sequence-based amplification (NASBA)".
840:
Collins, RA; Ko, LS; So, KL; Ellis, T; Lau, LT; Yu, AC (2002). "Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA".
71:
RNA template added to the reaction mixture, the first primer with the T7 promoter region on its 5' end attaches to its complementary site at the 3' end of the template.
125:
The NASBA technique has been used to develop rapid diagnostic tests for several pathogenic viruses with single-stranded RNA genomes, e.g. influenza A, zika virus,
294:
Lamb, Laura E.; Bartolone, Sarah N.; Tree, Maya O.; Conway, Michael J.; Rossignol, Julien; Smith, Christopher P.; Chancellor, Michael B. (December 2018).
39:
380:
237:
Reed, Adam J.; Connelly, Ryan P.; Williams, Allison; Tran, Maithi; Shim, Byoung-Shik; Choe, Hyeryun; Gerasimova, Yulia V. (March 2019).
163:
744:"Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum"
296:"Rapid Detection of Zika Virus in Urine Samples and Infected Mosquitos by Reverse Transcription-Loop-Mediated Isothermal Amplification"
875:
Collins, RA; Ko, LS; Fung, KY; Lau, LT; Xing, J; Yu, AC (2002). "A method to detect major serotypes of foot-and-mouth disease virus".
1057:"INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing"
154:
Recently, NASBA reaction with fluoresce, dipstick and next generation sequencing readout has been developed for COVID-19 diagnosis.
88:
destroys the RNA template from the DNA-RNA compound (RNAse H only destroys RNA in RNA-DNA hybrids, but not single-stranded RNA).
1008:"Nucleic acid sequence-based amplification with oligochromatography for detection of Trypanosoma brucei in clinical samples"
452:
427:
402:
79:
576:"Point-of-care diagnostic assay for the detection of Zika virus using the recombinase polymerase amplification method"
104:
Now a cyclic phase can begin similar to the previous steps. Here, however, the second primer first binds to the (-)RNA
94:
Reverse transcriptase again synthesizes another DNA strand from the attached primer resulting in double stranded DNA.
1122:
922:"Real-time NASBA detection of SARS-associated coronavirus and comparison with real-time reverse transcription-PCR"
116:
Exactly like step 6, the T7 polymerase binds to the promoter region to produce (-)RNA, and the cycle is complete.
52:
1055:
Wu, Qianxin; Suo, Chenqu; Brown, Tom; Wang, Tengyao; Teichmann, Sarah A.; Bassett, Andrew R. (February 2021).
574:
Vasileva Wand, Nadina I.; Bonney, Laura C.; Watson, Robert J.; Graham, Victoria; Hewson, Roger (August 2018).
884:
126:
791:
Arnaud-Barbe, Nadege; Cheynet
Sauvion, Valerie; Oriol, Guy; Mandrand, Bernard; Mallet, Francois (1998).
56:
664:
307:
1117:
889:
742:
Schneider, P; Wolters, L; Schoone, G; Schallig, H; Sillekens, P; Hermsen, R; Sauerwein, R (2005).
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556:
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376:
361:"Evaluation of Diagnostic Tests — Special Problems Introduced by DNA Amplification Procedures"
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24:
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250:
195:
130:
110:
RNAse H again degrades the RNA and the first primer binds to the now single stranded +(cDNA)
142:
668:
311:
113:
The reverse transcriptase now produces the complementary (-)DNA, creating a dsDNA duplex
1089:
1056:
1032:
1007:
948:
921:
608:
575:
336:
295:
271:
238:
184:"Characteristics and Applications of Nucleic Acid Sequence-Based Amplification (NASBA)"
898:
853:
817:
792:
768:
743:
1111:
720:
239:"Label-free pathogen detection by a deoxyribozyme cascade with visual signal readout"
560:
511:
223:
984:
692:
759:
360:
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Mugasa, CM; Laurent, T; Schoone, GJ; Kager, PA; Lubega, GW; Schallig, HD (2009).
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Keightley, MC; Sillekens, P; Schippers, W; Rinaldo, C; George, KS (2005).
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1023:
591:
183:
365:
Nucleic Acid
Amplification Technologies Application to Disease Diagnosis
91:
The second primer attaches to the 5' end of the (antisense) DNA strand.
85:
938:
676:
631:
655:
Compton, J (1991). "Nucleic acid sequence-based amplification".
531:. Methods in Molecular Biology. Vol. 28. pp. 253–260.
482:. Methods in Molecular Biology. Vol. 28. pp. 253–260.
134:
107:
The reverse transcriptase now produces a (+)cDNA/(-)RNA duplex.
51:
HIV-1 in patient sera. Although RNA can also be amplified by
182:
Deiman, Birgit; van Aarle, Pierre; Sillekens, Peter (2002).
42:(LAMP) is another isothermal amplification technique.
793:"Transcription of RNA templates by T7 RNA polymerase"
877:
367:, Boston, MA: Birkhäuser Boston, pp. 165–169,
78:strand extending the 3' end of the primer, moving
528:Nucleic acid sequence-based amplification (NASBA)
479:Nucleic acid sequence-based amplification (NASBA)
632:"PDB101: Molecule of the Month: RNA Polymerase"
74:Reverse transcriptase synthesizes the opposite
525:Malek, L.; Sooknanan, R.; Compton, J. (1994).
476:Malek, L.; Sooknanan, R.; Compton, J. (1994).
8:
67:Explained briefly, NASBA works as follows:
17:Nucleic acid sequence-based amplification,
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1031:
947:
937:
888:
816:
767:
607:
335:
270:
174:
40:Loop-mediated isothermal amplification
7:
164:Real-time polymerase chain reaction
14:
243:Sensors and Actuators B: Chemical
131:severe acute respiratory syndrome
1012:Journal of Clinical Microbiology
748:Journal of Clinical Microbiology
453:"Isothermal Amplification | NEB"
428:"Isothermal Amplification | NEB"
403:"Isothermal Amplification | NEB"
580:The Journal of General Virology
145:(HBoV) and also parasites like
985:10.1016/j.jviromet.2009.02.010
973:Journal of Virological Methods
842:Journal of Virological Methods
709:Journal of Virological Methods
1:
899:10.1016/S0006-291X(02)02178-2
854:10.1016/S0166-0934(02)00034-4
760:10.1128/JCM.43.1.402-405.2005
721:10.1016/0166-0934(91)90069-c
373:10.1007/978-1-4612-2454-9_12
926:Journal of Medical Virology
1139:
359:Schachter, Julius (1997),
320:10.1038/s41598-018-22102-5
537:10.1385/0-89603-254-x:253
488:10.1385/0-89603-254-x:253
255:10.1016/j.snb.2018.11.147
19:commonly referred to as
188:Molecular Biotechnology
82:along the RNA template.
1073:10.1126/sciadv.abe5054
809:10.1093/nar/26.15.3550
797:Nucleic Acids Research
451:Biolabs, New England.
426:Biolabs, New England.
401:Biolabs, New England.
127:foot-and-mouth disease
121:Clinical applications
57:reverse transcriptase
1024:10.1128/JCM.01430-08
592:10.1099/jgv.0.001083
669:1991Natur.350...91C
312:2018NatSR...8.3803L
200:10.1385/mb:20:2:163
300:Scientific Reports
148:Trypanosoma brucei
1123:Molecular biology
939:10.1002/jmv.20498
803:(15): 3550–3554.
382:978-1-4612-7543-5
101:in amplification.
98:T7 RNA polymerase
76:complementary DNA
25:molecular biology
23:, is a method in
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1061:Science Advances
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890:10.1.1.328.625
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663:(6313): 91–2.
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623:
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194:(2): 163–180.
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1018:(3): 630–5.
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932:(4): 602–8.
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786:
754:(1): 402–5.
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639:. Retrieved
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460:. Retrieved
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410:. Retrieved
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49:
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20:
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15:
457:www.neb.com
432:www.neb.com
407:www.neb.com
306:(1): 3803.
249:: 945–951.
139:coronavirus
1118:Amplifiers
1112:Categories
641:2020-11-15
462:2020-11-15
437:2020-11-15
412:2020-11-15
388:2020-11-15
170:References
31:Background
1081:2375-2548
885:CiteSeerX
600:1465-2099
545:1064-3745
496:1064-3745
328:2045-2322
263:0925-4005
208:1073-6085
63:Procedure
1099:33579697
1042:19116352
993:19428591
958:16254971
907:12237113
862:12008015
778:15635001
618:29897329
561:30720773
512:30720773
346:29491389
281:31462856
224:28712952
216:11876473
158:See also
80:upstream
55:using a
1090:7880595
1033:2650916
949:7167117
827:9671817
729:1726172
693:4304204
685:1706072
665:Bibcode
609:6171711
553:7509695
504:7509695
337:5830622
308:Bibcode
272:6713451
129:virus,
86:RNAse H
46:History
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557:S2CID
508:S2CID
220:S2CID
21:NASBA
1095:PMID
1077:ISSN
1038:PMID
989:PMID
954:PMID
903:PMID
858:PMID
823:PMID
774:PMID
725:PMID
681:PMID
614:PMID
596:ISSN
549:PMID
541:ISSN
500:PMID
492:ISSN
377:ISBN
342:PMID
324:ISSN
277:PMID
259:ISSN
212:PMID
204:ISSN
135:SARS
1085:PMC
1069:doi
1028:PMC
1020:doi
981:doi
977:158
944:PMC
934:doi
895:doi
881:297
850:doi
846:103
813:PMC
805:doi
764:PMC
756:doi
717:doi
673:doi
661:350
604:PMC
588:doi
533:doi
484:doi
369:doi
332:PMC
316:doi
267:PMC
251:doi
247:282
196:doi
53:PCR
1114::
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