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Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection.

Tong Y, Lemieux B, Kong H - BMC Biotechnol. (2011)

Bottom Line: The effect of combing all strategies was compared with that of the individual strategy.Some of them can be adjusted and applied to other formats of nucleic acid amplification.Furthermore, the strategies to improve the in vitro assays by maximally simulating the nature conditions may be useful in the general field of developing molecular assays.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioHelix Corp, Beverly, MA, USA. tong@biohelix.com

ABSTRACT

Background: In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).

Results: Multiple strategies were approached to improve the overall performance of the isothermal amplification: the restriction endonuclease-mediated DNA helicase homing, macromolecular crowding agents, and the optimization of reaction enzyme mix. The effect of combing all strategies was compared with that of the individual strategy. With all of above methods, we are able to detect 50 copies of Neisseria gonorrhoeae DNA in just 20 minutes of amplification using a nearly instrument-free detection platform (BEStâ„¢ cassette).

Conclusions: The strategies addressed in this proof-of-concept study are independent of expensive equipment, and are not limited to particular primers, targets or detection format. However, they make a large difference in assay performance. Some of them can be adjusted and applied to other formats of nucleic acid amplification. Furthermore, the strategies to improve the in vitro assays by maximally simulating the nature conditions may be useful in the general field of developing molecular assays. A new fast molecular assay for Neisseria gonorrhoeae has also been developed which has great potential to be used at point-of-care diagnostics.

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Schematic model of restriction endonucleases effects on HDA. Panel A.) Mechanism in vivo: The mismatch is recognized by MutS. MutS and MutL form complex to stimulate MutH which generates nick site of the DNA duplex near the mismatch position. UvrD (helicase) is then loaded, unwinds the DNA duplex at the nick, and extends toward the mismatch. Panel B.) Mechanism in vitro: Restriction endonucleases specifically cut the DNA duplex near the target sequence, generate blunt or 5' ss or 3' ss ends or nick site (if using nicking enzyme). UvrD is then loaded and unwinds the DNA duplex. Red lines represent target DNA sequence. Blue lines represent non-target DNA sequence.
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Figure 1: Schematic model of restriction endonucleases effects on HDA. Panel A.) Mechanism in vivo: The mismatch is recognized by MutS. MutS and MutL form complex to stimulate MutH which generates nick site of the DNA duplex near the mismatch position. UvrD (helicase) is then loaded, unwinds the DNA duplex at the nick, and extends toward the mismatch. Panel B.) Mechanism in vitro: Restriction endonucleases specifically cut the DNA duplex near the target sequence, generate blunt or 5' ss or 3' ss ends or nick site (if using nicking enzyme). UvrD is then loaded and unwinds the DNA duplex. Red lines represent target DNA sequence. Blue lines represent non-target DNA sequence.

Mentions: The helicase used in thermophilic HDA [7] belongs to the mismatch repair system in vivo. Figure 1A shows the mechanism of E. coli UvrD mismatch repair system, which is the most understood system [10], and most likely resembles the mechanism through which thermophilic helicase operates. The E. coli system requires multiple accessory proteins (e.g. at least mutS, mutL and mutH) to generate nicks near the mismatch sites and then load the UvrD, which has high affinity in binding the ends of nucleic acid molecules [7,11]. However, from a manufacturing perspective, purifying so many accessory proteins to perform in vitro amplifications would be too costly. Although the nucleic acid substrates input into in vitro amplification reactions are sheared or nicked during the extraction and purification steps, the DNA ends generated by this process are randomly distributed along the nucleic acid substrates and are not target specific and thus not to aid HDA. As shown in Table 1 the Tt (Threshold time, is defined as the number of detection cycles required for the fluorescent signal to cross the threshold. It is a similar definition as cycle threshold for real-time PCR. The only difference is that the cycle in real-time PCR is thermal cycle. In order to compare the assay speed and robustness, the Tt value is converted to minutes by 1 Tt = 2 minutes. The set-up details of detection cycle are described in the Methods section) values of HDA amplification on low copy number targets are distributed widely over 3-6 minutes. We hypothesize that reactions with low copy number of targets benefit more obviously from a proximal nick or double strand break in the template nucleic acid than those with high copy number of targets. Because loading helicases near the rare amount of target region is a random event with low chance.


Multiple strategies to improve sensitivity, speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection.

Tong Y, Lemieux B, Kong H - BMC Biotechnol. (2011)

Schematic model of restriction endonucleases effects on HDA. Panel A.) Mechanism in vivo: The mismatch is recognized by MutS. MutS and MutL form complex to stimulate MutH which generates nick site of the DNA duplex near the mismatch position. UvrD (helicase) is then loaded, unwinds the DNA duplex at the nick, and extends toward the mismatch. Panel B.) Mechanism in vitro: Restriction endonucleases specifically cut the DNA duplex near the target sequence, generate blunt or 5' ss or 3' ss ends or nick site (if using nicking enzyme). UvrD is then loaded and unwinds the DNA duplex. Red lines represent target DNA sequence. Blue lines represent non-target DNA sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104364&req=5

Figure 1: Schematic model of restriction endonucleases effects on HDA. Panel A.) Mechanism in vivo: The mismatch is recognized by MutS. MutS and MutL form complex to stimulate MutH which generates nick site of the DNA duplex near the mismatch position. UvrD (helicase) is then loaded, unwinds the DNA duplex at the nick, and extends toward the mismatch. Panel B.) Mechanism in vitro: Restriction endonucleases specifically cut the DNA duplex near the target sequence, generate blunt or 5' ss or 3' ss ends or nick site (if using nicking enzyme). UvrD is then loaded and unwinds the DNA duplex. Red lines represent target DNA sequence. Blue lines represent non-target DNA sequence.
Mentions: The helicase used in thermophilic HDA [7] belongs to the mismatch repair system in vivo. Figure 1A shows the mechanism of E. coli UvrD mismatch repair system, which is the most understood system [10], and most likely resembles the mechanism through which thermophilic helicase operates. The E. coli system requires multiple accessory proteins (e.g. at least mutS, mutL and mutH) to generate nicks near the mismatch sites and then load the UvrD, which has high affinity in binding the ends of nucleic acid molecules [7,11]. However, from a manufacturing perspective, purifying so many accessory proteins to perform in vitro amplifications would be too costly. Although the nucleic acid substrates input into in vitro amplification reactions are sheared or nicked during the extraction and purification steps, the DNA ends generated by this process are randomly distributed along the nucleic acid substrates and are not target specific and thus not to aid HDA. As shown in Table 1 the Tt (Threshold time, is defined as the number of detection cycles required for the fluorescent signal to cross the threshold. It is a similar definition as cycle threshold for real-time PCR. The only difference is that the cycle in real-time PCR is thermal cycle. In order to compare the assay speed and robustness, the Tt value is converted to minutes by 1 Tt = 2 minutes. The set-up details of detection cycle are described in the Methods section) values of HDA amplification on low copy number targets are distributed widely over 3-6 minutes. We hypothesize that reactions with low copy number of targets benefit more obviously from a proximal nick or double strand break in the template nucleic acid than those with high copy number of targets. Because loading helicases near the rare amount of target region is a random event with low chance.

Bottom Line: The effect of combing all strategies was compared with that of the individual strategy.Some of them can be adjusted and applied to other formats of nucleic acid amplification.Furthermore, the strategies to improve the in vitro assays by maximally simulating the nature conditions may be useful in the general field of developing molecular assays.

View Article: PubMed Central - HTML - PubMed

Affiliation: BioHelix Corp, Beverly, MA, USA. tong@biohelix.com

ABSTRACT

Background: In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).

Results: Multiple strategies were approached to improve the overall performance of the isothermal amplification: the restriction endonuclease-mediated DNA helicase homing, macromolecular crowding agents, and the optimization of reaction enzyme mix. The effect of combing all strategies was compared with that of the individual strategy. With all of above methods, we are able to detect 50 copies of Neisseria gonorrhoeae DNA in just 20 minutes of amplification using a nearly instrument-free detection platform (BEStâ„¢ cassette).

Conclusions: The strategies addressed in this proof-of-concept study are independent of expensive equipment, and are not limited to particular primers, targets or detection format. However, they make a large difference in assay performance. Some of them can be adjusted and applied to other formats of nucleic acid amplification. Furthermore, the strategies to improve the in vitro assays by maximally simulating the nature conditions may be useful in the general field of developing molecular assays. A new fast molecular assay for Neisseria gonorrhoeae has also been developed which has great potential to be used at point-of-care diagnostics.

Show MeSH
Related in: MedlinePlus