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LAMP – a powerful and flexible tool for monitoring microbial pathogens
Update
Trends in Parasitology Vol.25 No.11
Letters
LAMP – a powerful and flexible tool for monitoring microbial pathogens Panagiotis Karanis1 and Jerry Ongerth2 1
Medical School, University of Cologne, Department of Anatomy, Institute II, Medical and Molecular Parasitology Laboratory, Centre of Anatomy, Institute II, Kerpener Str. 62, 50937 Cologne, Germany 2 Civil, Mining and Environmental Engineering, University of Wollongong, Wollongong NSW 2522 Australia
Loop-mediated isothermal amplification (LAMP) is one of the nucleic acid amplifications tests (NATs) available for organism identification applications in various fields such as infection diagnosis. It has been commonly described as a novel method, yet over 250 publications have appeared in less than 10 years since its original description [1]. LAMP has been applied to produce highly specific and sensitive amplification of DNA or RNA from virtually every corner of the biological world, including prokaryotes and eukaryotes, plant and animal tissue. Among published studies, the majority are descriptions of the development and application of LAMP to detect human pathogens including virus, bacteria, protozoa and fungi [2]. Briefly, LAMP employs four primers recognizing six independent sequences of the selected target gene for initiation, with four sequences subsequently used for amplification and visualisation of the amplified product. It uses a robust polymerase (BST) to amplify target DNA (or RNA by inclusion of reverse transcriptase) proceeding to an autocycling strand displacement mechanism, at a constant temperature, producing detectable product in approximately 1 h [1]. Defining features of LAMP identified through its relatively short period of development include the following: it is highly selective (i.e. able to distinguish between organism subtypes) and highly sensitive, often demonstrated to amplify from a single copy or from a single organism. It is robust, with reagents stable at ambient temperature for up to 2 weeks [3], and consistently insensitive to extraneous nucleic acids or interference from sample or media components that are problematic for other NATs. The procedure is rapid and is able to amplify from a single copy to 109 in 1 h at constant temperature, typically in the range of 60–708 C. Requirements for sample processing and LAMP application are relatively simple, not requiring high technical skill or sophisticated equipment. Issues such as contamination and post-amplification handling, spatial separation of work areas and direct comparison of LAMP with real-time PCR continue to be elucidated by new and innovative work. Further prospective validation in field settings will be important to broadening LAMP application and acceptance [4]. In studies, particularly over the past 5 years, investigators have used different approaches to gain higher levels of resolution among closely related targets. In studies to identify any Cryptosporidium species oocysts Corresponding author: Karanis, P. ([email protected]).
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in environmental waters or in faecal samples, species level C. parvum target was used [5]. In subsequent studies, genome regions were targeted that are established as specific to combinations of species [6]. Elsewhere, different primer sets were used to distinguish between four species of trypanosomes: Trypanosoma brucei gambiense, T. congolense, T. cruzi and T. evansi [3]. A multiplex LAMP combining four primer sets in a single LAMP assay mixture was used to distinguish two species of Babesia (B. bovis and B. bigemina), with separation provided by subsequent EcoR1 digestion and electrophoresis [7]. However, restriction digestion after LAMP presents a risk for contamination that must be avoided. Using a seven-primer approach in a single reaction tube, RNAs of three culture-adapted hepatitis A virus strains in the subgenotypes IA, IB and IIIB were identified [8]. The ability of LAMP to distinguish between organisms differing by only a single nucleotide polymorphism has been utilised to identify closely related human herpes virus types 6A and B [9]. Detection limits for virus in specimen and media samples has often been reported as a single copy. For example, a LAMP for HIV amplified from 10–100 copies per reaction to detectable product in finger stick blood samples [10,11]. Hepatitis B virus was detected by a RT-LAMP using a fluorogenic endpoint at the equivalent of 4.4 copies per ml, providing good agreement with real-time PCR applied to 400 clinical samples [12]. Similar sensitivity accompanying specificity has been observed, for example in bacterial LAMPs (e.g. Mycobacterium tuberculosis [12]) and LAMPs directed to protozoa (e.g. Plasmodium [13]). The increasing application of LAMP to nucleic acid extracts of unpurified samples or even to samples without nucleic acid extraction demonstrates its general insensitivity to extraneous materials other than the target. Arbovirus and Plasmodium (oocyst and sporozoite or other stage DNA) have been identified in whole mosquitoes processed only by tissue grinding [2,14]. Hepatitis A virus, Cryptosporidium oocyst and Toxoplasma oocyst DNA have been detected efficiently in crude faecal nucleic acid extracts. The specificity and sensitivity of detection do not seem to be impaired by LAMP processing conditions or sample type [2,15], including whole blood, boiled or cardprocessed blood, serum, sputum and crudely processed tissue samples. However, regarding Plasmodium spp. detection, malaria diagnosis by LAMP requires further prospective validation to establish sensitivity. Further research towards optimising and simplifying template
Update production methods will also be important. LAMP is an emerging technology in the field of parasitology and further efforts in ongoing work should soon provide more comparative sensitivity results in the diagnosis of parasitic diseases. The potential for broad applicability of LAMP derives from the characteristics described above. The rapid development of many and varied applications are a product of these characteristics. Continued development will progress based on the ingenuity of individual investigators and clinicians and on opportunities to utilise LAMP characteristics to solve organism diagnostic identification problems. References 1 Notomi, T. et al. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, 12 2 Mori, Y. and Notomi, T. (2009) Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J. Infect. Chemother. 15, 62–69 3 Thekisoe, O. et al. (2009) Stability of loop mediated isothermal amplification (LAMP) reagents and its amplification efficiency on crude trypanosome DNA templates. J. Vet. Med. Sci. 71, 471–475 4 Paris, D.H. et al. (2008) Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification. Trans. R. Soc. Trop. Med. Hyg. 102, 1239–1246 5 Karanis, P. et al. (2007) Development and preliminary evaluation of a loop-mediated isothermal amplification procedure for sensitive detection of Cryptosporidium oocysts in fecal and water samples. Appl. Environ. Microbiol. 73, 5660–5662 6 Bakheit, M. et al. (2008) Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated
Trends in Parasitology
Vol.25 No.11
isothermal DNA amplification and confirmation of generated LAMP products by sequencing. Vet. Parasitol. 25, 11–22 7 Iseki, H. et al. (2007) Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites. J. Microbiol. Methods 71, 281–287 8 Yoneyama, T. et al. (2007) Rapid and real-time detection of hepatitis A virus by reverse transcription loop-mediated isothermal amplification assay. J. Virol. Methods 145, 162–168 9 Ihira, M. et al. (2008) Loop-mediated isothermal amplification for discriminating between human herpes virus 6 A and B. J. Virol. Methods 154, 223–225 10 Curtis, K.A. et al. (2008) Rapid detection of HIV-1 b reversetranscription loop-mediated isothermal amplification (RT-LAMP). J. Virol. Methods 151, 264–270 11 Cai, T. et al. (2008) Development and evaluation of real-time loopmediated isothermal amplification for hepatitis B virus DNA quantification: a new tool for HBV management. J. Clin. Virol. 41, 270–276 12 Lee, M.F. et al. (2009) Evaluation of reverse loop-mediated isothermal amplification in conjunction with ELISA-hybridization assay for molecular detection of Mycobacterium tuberculosis. J. Microbiol. Methods 76, 174–180 13 Poon, L.L. et al. (2006) Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin. Chem. 52, 303–306 14 Aonuma, H. et al. (2008) Rapid identification of Plasmodium-carrying mosquitoes using loop-mediated isothermal amplification. Biochem. Biophys. Res. Commun. 376, 671–676 15 Kaneko, H. et al. (2007) Tolerance of loop-mediated isothermal amplification to the culture medium and biological substances. J. Biochem. Biophys. Methods 70, 499–501 1471-4922/$ – see front matter ß 2009 Published by Elsevier Ltd. doi:10.1016/j.pt.2009.07.010 Available online 4 September 2009
499
Trends in Parasitology Vol.25 No.11
Letters
LAMP – a powerful and flexible tool for monitoring microbial pathogens Panagiotis Karanis1 and Jerry Ongerth2 1
Medical School, University of Cologne, Department of Anatomy, Institute II, Medical and Molecular Parasitology Laboratory, Centre of Anatomy, Institute II, Kerpener Str. 62, 50937 Cologne, Germany 2 Civil, Mining and Environmental Engineering, University of Wollongong, Wollongong NSW 2522 Australia
Loop-mediated isothermal amplification (LAMP) is one of the nucleic acid amplifications tests (NATs) available for organism identification applications in various fields such as infection diagnosis. It has been commonly described as a novel method, yet over 250 publications have appeared in less than 10 years since its original description [1]. LAMP has been applied to produce highly specific and sensitive amplification of DNA or RNA from virtually every corner of the biological world, including prokaryotes and eukaryotes, plant and animal tissue. Among published studies, the majority are descriptions of the development and application of LAMP to detect human pathogens including virus, bacteria, protozoa and fungi [2]. Briefly, LAMP employs four primers recognizing six independent sequences of the selected target gene for initiation, with four sequences subsequently used for amplification and visualisation of the amplified product. It uses a robust polymerase (BST) to amplify target DNA (or RNA by inclusion of reverse transcriptase) proceeding to an autocycling strand displacement mechanism, at a constant temperature, producing detectable product in approximately 1 h [1]. Defining features of LAMP identified through its relatively short period of development include the following: it is highly selective (i.e. able to distinguish between organism subtypes) and highly sensitive, often demonstrated to amplify from a single copy or from a single organism. It is robust, with reagents stable at ambient temperature for up to 2 weeks [3], and consistently insensitive to extraneous nucleic acids or interference from sample or media components that are problematic for other NATs. The procedure is rapid and is able to amplify from a single copy to 109 in 1 h at constant temperature, typically in the range of 60–708 C. Requirements for sample processing and LAMP application are relatively simple, not requiring high technical skill or sophisticated equipment. Issues such as contamination and post-amplification handling, spatial separation of work areas and direct comparison of LAMP with real-time PCR continue to be elucidated by new and innovative work. Further prospective validation in field settings will be important to broadening LAMP application and acceptance [4]. In studies, particularly over the past 5 years, investigators have used different approaches to gain higher levels of resolution among closely related targets. In studies to identify any Cryptosporidium species oocysts Corresponding author: Karanis, P. ([email protected]).
498
in environmental waters or in faecal samples, species level C. parvum target was used [5]. In subsequent studies, genome regions were targeted that are established as specific to combinations of species [6]. Elsewhere, different primer sets were used to distinguish between four species of trypanosomes: Trypanosoma brucei gambiense, T. congolense, T. cruzi and T. evansi [3]. A multiplex LAMP combining four primer sets in a single LAMP assay mixture was used to distinguish two species of Babesia (B. bovis and B. bigemina), with separation provided by subsequent EcoR1 digestion and electrophoresis [7]. However, restriction digestion after LAMP presents a risk for contamination that must be avoided. Using a seven-primer approach in a single reaction tube, RNAs of three culture-adapted hepatitis A virus strains in the subgenotypes IA, IB and IIIB were identified [8]. The ability of LAMP to distinguish between organisms differing by only a single nucleotide polymorphism has been utilised to identify closely related human herpes virus types 6A and B [9]. Detection limits for virus in specimen and media samples has often been reported as a single copy. For example, a LAMP for HIV amplified from 10–100 copies per reaction to detectable product in finger stick blood samples [10,11]. Hepatitis B virus was detected by a RT-LAMP using a fluorogenic endpoint at the equivalent of 4.4 copies per ml, providing good agreement with real-time PCR applied to 400 clinical samples [12]. Similar sensitivity accompanying specificity has been observed, for example in bacterial LAMPs (e.g. Mycobacterium tuberculosis [12]) and LAMPs directed to protozoa (e.g. Plasmodium [13]). The increasing application of LAMP to nucleic acid extracts of unpurified samples or even to samples without nucleic acid extraction demonstrates its general insensitivity to extraneous materials other than the target. Arbovirus and Plasmodium (oocyst and sporozoite or other stage DNA) have been identified in whole mosquitoes processed only by tissue grinding [2,14]. Hepatitis A virus, Cryptosporidium oocyst and Toxoplasma oocyst DNA have been detected efficiently in crude faecal nucleic acid extracts. The specificity and sensitivity of detection do not seem to be impaired by LAMP processing conditions or sample type [2,15], including whole blood, boiled or cardprocessed blood, serum, sputum and crudely processed tissue samples. However, regarding Plasmodium spp. detection, malaria diagnosis by LAMP requires further prospective validation to establish sensitivity. Further research towards optimising and simplifying template
Update production methods will also be important. LAMP is an emerging technology in the field of parasitology and further efforts in ongoing work should soon provide more comparative sensitivity results in the diagnosis of parasitic diseases. The potential for broad applicability of LAMP derives from the characteristics described above. The rapid development of many and varied applications are a product of these characteristics. Continued development will progress based on the ingenuity of individual investigators and clinicians and on opportunities to utilise LAMP characteristics to solve organism diagnostic identification problems. References 1 Notomi, T. et al. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, 12 2 Mori, Y. and Notomi, T. (2009) Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J. Infect. Chemother. 15, 62–69 3 Thekisoe, O. et al. (2009) Stability of loop mediated isothermal amplification (LAMP) reagents and its amplification efficiency on crude trypanosome DNA templates. J. Vet. Med. Sci. 71, 471–475 4 Paris, D.H. et al. (2008) Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification. Trans. R. Soc. Trop. Med. Hyg. 102, 1239–1246 5 Karanis, P. et al. (2007) Development and preliminary evaluation of a loop-mediated isothermal amplification procedure for sensitive detection of Cryptosporidium oocysts in fecal and water samples. Appl. Environ. Microbiol. 73, 5660–5662 6 Bakheit, M. et al. (2008) Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated
Trends in Parasitology
Vol.25 No.11
isothermal DNA amplification and confirmation of generated LAMP products by sequencing. Vet. Parasitol. 25, 11–22 7 Iseki, H. et al. (2007) Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites. J. Microbiol. Methods 71, 281–287 8 Yoneyama, T. et al. (2007) Rapid and real-time detection of hepatitis A virus by reverse transcription loop-mediated isothermal amplification assay. J. Virol. Methods 145, 162–168 9 Ihira, M. et al. (2008) Loop-mediated isothermal amplification for discriminating between human herpes virus 6 A and B. J. Virol. Methods 154, 223–225 10 Curtis, K.A. et al. (2008) Rapid detection of HIV-1 b reversetranscription loop-mediated isothermal amplification (RT-LAMP). J. Virol. Methods 151, 264–270 11 Cai, T. et al. (2008) Development and evaluation of real-time loopmediated isothermal amplification for hepatitis B virus DNA quantification: a new tool for HBV management. J. Clin. Virol. 41, 270–276 12 Lee, M.F. et al. (2009) Evaluation of reverse loop-mediated isothermal amplification in conjunction with ELISA-hybridization assay for molecular detection of Mycobacterium tuberculosis. J. Microbiol. Methods 76, 174–180 13 Poon, L.L. et al. (2006) Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin. Chem. 52, 303–306 14 Aonuma, H. et al. (2008) Rapid identification of Plasmodium-carrying mosquitoes using loop-mediated isothermal amplification. Biochem. Biophys. Res. Commun. 376, 671–676 15 Kaneko, H. et al. (2007) Tolerance of loop-mediated isothermal amplification to the culture medium and biological substances. J. Biochem. Biophys. Methods 70, 499–501 1471-4922/$ – see front matter ß 2009 Published by Elsevier Ltd. doi:10.1016/j.pt.2009.07.010 Available online 4 September 2009
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