Loop mediated isothermal amplification (LAMP),Is it a cost-effective alternative for PCR?

 

 

By

Dr. K.M. Gunasekera
Senior Lecturer,
Department of Microbiology
Faculty of Medical Sciences
University of Sri Jayewardenepura

 

Loop mediated isothermal amplification (LAMP) is a novel technique first developed in the year 2000 by Notomi et al and further improved by Nagamine et al.1,2 The simplicity of the procedure belies the complex chemical and molecular processes that are involved in the reaction. A detailed explanation of the mechanism is beyond the scope of this article, but very briefly, it involves six primers targeting eight different areas of the target sequence, which are amplified by a DNA polymerase under isothermal (usually 60-65oC) conditions. The cycling reaction results in 109 copies of the target in less than hour (Figure 1).1 LAMP assays have been developed for all types of pathogens and lately for genotyping as well.3

 The most appealing feature of LAMP assays is the short turnaround time for results (incubation time 30-60 minutes). The need for thermocyclers is eliminated as incubation is done in a water bath, simplifying laboratory requirements. Commercial extraction kits though convenient for use, increases the cost of polymerase chain reactions (PCR). LAMP is less susceptible to inhibition by proteins and other factors that inhibit (PCR). Therefore, crude extraction methods of DNA/RNA such as boiling or unprocessed specimens have been successfully used with LAMP,4  thus reducing the cost of LAMP significantly.

Results of PCR are detected by gel electrophoresis or fluorescence signals in real-time thermocyclers. LAMP results can be read using visual indicators which are less expensive and time saving.  Advances in LAMP technique have eliminated the need for expensive turbidity meters by the discovery of visual indicators. Visual indicators such as intercalating dyes (e.g. Sybr® Green ), magnesium chelators (e.g. hydroxyl naphthol blue) and pH indicators (e.g. Phenol Red, Neutral Red) have led to the development of cheaper LAMP assays and lateral flow tests for point-of-care testing.4,5 LAMP is more highly sensitive and specific than conventional PCR and as good as real-time PCR.6 However, increased sensitivity of LAMP necessitates rigorous precautions to avoid contamination by aerosols.

Having four to six primers in LAMP increases the specificity of the test, but at the same time it makes development of single tube multiplex LAMP assays difficult. Currently the most expensive component is the LAMP polymerase (more expensive than Taq polymerase). But advances in biochemical research continues to uncover LAMP polymerases that are cheaper. Most investigators prefer commercially available master-mixes such as LoopAmp® DNA amplification kits (Eiken Chemical Co. Ltd, Japan), which are expensive. In-house preparation of master-mixes or use of polymerase buffer is far more cost effective. The following example illustrates the significant difference in costs for Chlamydia trachomatis infection detection by LAMP and real-time PCR. Reagents for a LAMP assay4 using crude extracts of endocervical specimens would cost approximately Rs. 350.00 per test, whereas for a single real-time PCR (Qiagen) test it would be around Rs. 3200.00.

The current covid-19 pandemic has shown us the importance of having rapid and inexpensive testing methods. LAMP is an attractive alternative to PCR for resource limited laboratories as it is cheaper, rapid, easy-to-use and does not require  sophisticated instruments. Local researchers should channel their efforts into developing more LAMP assays for infections that currently require expensive and labour intensive diagnostic methods.

Figure 1. Schematic representation of the mechanism of LAMP

(Courtesy: Notomi et al, Nucleic Acids Research, 2000, Vol. 28, No. 12)

 

References 

  1. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000 Jun 15;28(12):e63.
  2. Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002 Jun;16(3):223–9
  3. Gill P and Amree AH. AS-LAMP: A new and alternative method for genotyping. Avicenna J Med Biotechnol 2020;12(1):2-8
  4. Somboonna N, Choopara I, Arunrut N, Sukhonpan K, Sayasathid J, Dean D, et al. Rapid and sensitive detection of Chlamydia trachomatis sexually transmitted infections in resource-constrained settings in Thailand at the point-of-care. PLoS Negl Trop Dis 2018; 12(12): e0006900
  5. Scott AT, Layne TR, O'Connell K, Tanner NA, James P. Landers JP. Comparative evaluation and quantitative analysis of loop-mediated isothermal amplification indicators. Chem.2020;92:13343–53
  6. Hagiwara M, Sasaki H, Matsuo K, Honda M, Kawase M, Nakagawa Loop-mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18 J Med Virol. 2007;79:605-15.

Author: Roshan jayasuriya