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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 (BESt™ 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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Related in: MedlinePlus

Rapid amplification and detection of Neisseria gonorrhea genomic DNA. Panel A) Fluorescence monitoring in real-time is shown as semi-log plot of fluorescence intensity versus Tt number where each cycle is 1 minute-long. The copy numbers of input template are labeled on the figure. Panel B) Type II BESt™ cassette detection of 5e4 copies/assay (strips 1-2), 500 copies/assay (strips 3-4), 50 copies/assay (strips 5-7), and non template control (NTC) (strips 8-9). The signal from NTC (either in panel A or B) is the amplification signal from the internal control sequence. The IC sequence shares the same primer sequence with the detected target gene of Neisseria gonorrhoeae, just with different internal nucleic acid sequence between the primers. The IC sequence is cloned into a plasmid. When a reaction is set-up, the IC plasmid is premixed with all of the other reaction components (except the target DNA or tested clinical samples) to prepare the master mix of the assay. When there is no target template of Neisseria gonorrhoeae (or negative clinical samples) in the assay, the IC sequence is amplified. Because the different internal sequence between the IC sequence and the target sequence of Neisseria gonorrhoeae, different probes are designed and included in the reaction. This can be differentiated from T line (Test line: for the detection of target DNA amplification products by target probe NGP) and C line (Control line: for the detection of internal control amplification products by IC probe NGICP) in Panel B.
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Figure 3: Rapid amplification and detection of Neisseria gonorrhea genomic DNA. Panel A) Fluorescence monitoring in real-time is shown as semi-log plot of fluorescence intensity versus Tt number where each cycle is 1 minute-long. The copy numbers of input template are labeled on the figure. Panel B) Type II BESt™ cassette detection of 5e4 copies/assay (strips 1-2), 500 copies/assay (strips 3-4), 50 copies/assay (strips 5-7), and non template control (NTC) (strips 8-9). The signal from NTC (either in panel A or B) is the amplification signal from the internal control sequence. The IC sequence shares the same primer sequence with the detected target gene of Neisseria gonorrhoeae, just with different internal nucleic acid sequence between the primers. The IC sequence is cloned into a plasmid. When a reaction is set-up, the IC plasmid is premixed with all of the other reaction components (except the target DNA or tested clinical samples) to prepare the master mix of the assay. When there is no target template of Neisseria gonorrhoeae (or negative clinical samples) in the assay, the IC sequence is amplified. Because the different internal sequence between the IC sequence and the target sequence of Neisseria gonorrhoeae, different probes are designed and included in the reaction. This can be differentiated from T line (Test line: for the detection of target DNA amplification products by target probe NGP) and C line (Control line: for the detection of internal control amplification products by IC probe NGICP) in Panel B.

Mentions: In order to test the optimal performance of the assay and the feasibility for type II BESt™ cassette detection after 20-minutes amplification, the reaction was set up with the final conditions as: standard HDA condition with addition of 4× IsoAmp III, 10 μL of 40% Ficoll 400, 5 units of MboI and 1e5 copies of internal control (IC) DNA. The purpose of including internal control DNA in each clinical assay is to monitor the potential inhibitors from clinical specimens which might generate false negative results. Generally, one kind of detection platform is used for each assay (either real-time based detection or BESt™ cassette based detection). However, in order to demonstrate that the improved Tt value (smaller number) also indicates that the shortened amplification time required for end-point detection, two detection methods were used at the same time in this study. Real-time detection dye (EvaGreen® and ROX) was included in the assay to evaluate the speed by Tt values, and the probes for cassette detection were also included in the reaction to verify the detection performance by cassette (as described in the Methods section). Assays were incubated in the ABI 7300 with the modified program to monitor the improved speed: 20 cycles of 66°C for 5 seconds, and 65°C for 55 seconds with data collection and fluorescence signal being collected at the end of each cycle (1 Tt number = 1 minute). After 20 minutes (20 cycles), the reaction tubes were immediately placed into type II BESt™ cassettes for detection of reaction products according to the package insert supplied by the vendor [9]. The results are shown in Figure 3. The Tt was less than 15 minutes for both 50 copies of target and NTC (where the internal control sequence was amplified in the absence of target) (Figure 3A). And the positive test lines were shown on the strips from the 50000, 500 and 50 copies of NG genomic DNA input. The control lines were shown on the strips from the non NG template control, where the amplified internal control sequence was detected here (Figure 3B). The data also demonstrated that the improvements of speed and robustness are not depended on particular detection format.


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)

Rapid amplification and detection of Neisseria gonorrhea genomic DNA. Panel A) Fluorescence monitoring in real-time is shown as semi-log plot of fluorescence intensity versus Tt number where each cycle is 1 minute-long. The copy numbers of input template are labeled on the figure. Panel B) Type II BESt™ cassette detection of 5e4 copies/assay (strips 1-2), 500 copies/assay (strips 3-4), 50 copies/assay (strips 5-7), and non template control (NTC) (strips 8-9). The signal from NTC (either in panel A or B) is the amplification signal from the internal control sequence. The IC sequence shares the same primer sequence with the detected target gene of Neisseria gonorrhoeae, just with different internal nucleic acid sequence between the primers. The IC sequence is cloned into a plasmid. When a reaction is set-up, the IC plasmid is premixed with all of the other reaction components (except the target DNA or tested clinical samples) to prepare the master mix of the assay. When there is no target template of Neisseria gonorrhoeae (or negative clinical samples) in the assay, the IC sequence is amplified. Because the different internal sequence between the IC sequence and the target sequence of Neisseria gonorrhoeae, different probes are designed and included in the reaction. This can be differentiated from T line (Test line: for the detection of target DNA amplification products by target probe NGP) and C line (Control line: for the detection of internal control amplification products by IC probe NGICP) in Panel B.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Rapid amplification and detection of Neisseria gonorrhea genomic DNA. Panel A) Fluorescence monitoring in real-time is shown as semi-log plot of fluorescence intensity versus Tt number where each cycle is 1 minute-long. The copy numbers of input template are labeled on the figure. Panel B) Type II BESt™ cassette detection of 5e4 copies/assay (strips 1-2), 500 copies/assay (strips 3-4), 50 copies/assay (strips 5-7), and non template control (NTC) (strips 8-9). The signal from NTC (either in panel A or B) is the amplification signal from the internal control sequence. The IC sequence shares the same primer sequence with the detected target gene of Neisseria gonorrhoeae, just with different internal nucleic acid sequence between the primers. The IC sequence is cloned into a plasmid. When a reaction is set-up, the IC plasmid is premixed with all of the other reaction components (except the target DNA or tested clinical samples) to prepare the master mix of the assay. When there is no target template of Neisseria gonorrhoeae (or negative clinical samples) in the assay, the IC sequence is amplified. Because the different internal sequence between the IC sequence and the target sequence of Neisseria gonorrhoeae, different probes are designed and included in the reaction. This can be differentiated from T line (Test line: for the detection of target DNA amplification products by target probe NGP) and C line (Control line: for the detection of internal control amplification products by IC probe NGICP) in Panel B.
Mentions: In order to test the optimal performance of the assay and the feasibility for type II BESt™ cassette detection after 20-minutes amplification, the reaction was set up with the final conditions as: standard HDA condition with addition of 4× IsoAmp III, 10 μL of 40% Ficoll 400, 5 units of MboI and 1e5 copies of internal control (IC) DNA. The purpose of including internal control DNA in each clinical assay is to monitor the potential inhibitors from clinical specimens which might generate false negative results. Generally, one kind of detection platform is used for each assay (either real-time based detection or BESt™ cassette based detection). However, in order to demonstrate that the improved Tt value (smaller number) also indicates that the shortened amplification time required for end-point detection, two detection methods were used at the same time in this study. Real-time detection dye (EvaGreen® and ROX) was included in the assay to evaluate the speed by Tt values, and the probes for cassette detection were also included in the reaction to verify the detection performance by cassette (as described in the Methods section). Assays were incubated in the ABI 7300 with the modified program to monitor the improved speed: 20 cycles of 66°C for 5 seconds, and 65°C for 55 seconds with data collection and fluorescence signal being collected at the end of each cycle (1 Tt number = 1 minute). After 20 minutes (20 cycles), the reaction tubes were immediately placed into type II BESt™ cassettes for detection of reaction products according to the package insert supplied by the vendor [9]. The results are shown in Figure 3. The Tt was less than 15 minutes for both 50 copies of target and NTC (where the internal control sequence was amplified in the absence of target) (Figure 3A). And the positive test lines were shown on the strips from the 50000, 500 and 50 copies of NG genomic DNA input. The control lines were shown on the strips from the non NG template control, where the amplified internal control sequence was detected here (Figure 3B). The data also demonstrated that the improvements of speed and robustness are not depended on particular detection format.

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 (BESt™ 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