Nucleic acid amplification is a valuable molecular tool not only in basic research but also in applicationoriented fields, such as clinical medicine development, infectious diseases diagnosis、gene cloning and industrial quality control etc. Several amplification methods have been developed already, such as polymerase chain reaction (PCR) (19), self-sustained sequence replication (3SR) (6), nucleic acid sequencebased amplification (NASBA) (2), strand displacement amplification (SDA) (22) and rolling circle amplification (RCA) (14) etc. Most of these methods can amplify targets by many magnitudes of order using its own particular mechanism to re-initiate new rounds of DNA synthesis, but still have many shortcomings, including the requirement of precision instruments and elaborate methods for product detection. LAMP is a novel nucleic acid amplification method developed by Notomi et al, which amplifies DNA with high specificity, sensitivity and rapidity under isothermal condition using a set of four specially designed primers and a Bst DNA polymerase (17). In this article, we overview the current status of LAMP and recent developments associated with the method.
Since the development of LAMP in 2000, many modifications and improvements have been reported.
LAMP product was previously detected by agarose gel electrophoresis and visualized by staining with ethidium bromide, which is either time consuming and does not meet the requirement of field tests. However, it was observed that a large amount of white precipitate was produced during the LAMP amplification (Fig. 2). Accordingly, Mori et al. clarified the cause of the precipitate production and used this to establish a new method for detection of LAMP products (15). Their method is based on the observation that the increase of the precipitate correlates with the amount of DNA synthesized and real-time monitoring of the LAMP reaction was achieved by measurement of turbidity.
Figure 2. White precipitate preduced in LAMP reaction. 1. Negtive control; 2. Positive LAMP reaction.
Nagamine et al. developed a method that could accelerate the LAMP reaction by using additional primers, termed loop primers (18). Loop primers hybridize to the stem-loops, with the exception of the loops that are hybridized by the inner primers, and prime strand displacement DNA synthesis. They found that this method could halve the reaction time to facilitated rapid application of the LAMP process. In addition, amplification could occur from 103 copies of the target using the loop primers while under the absence of loop primers, the amplification required more than 104copies, which suggested that LAMP reaction with the loop primers provided higher sensitivity and efficiency.
Many reports have described LAMP assay as being less affected in terms of sensitivity by the presence of inhibitory substances than PCR. Kaneko et al. have performed a detailed evaluation of the tolerance of LAMP against a culture medium and certain biological substances (11). They used the herpes simplex virus type (HSV) diluted with Minimum Essential Medium (MEM) as sample material. The results showed that HSV DNA was detected at a 10-5 dilution in both samples with and without DNA extraction, indicating that MEM did not affect the sensitivity of LAMP. Other substances were all tested including PBS, Serum, Plasma, Urine, and Vitreous (Table 1), which showed that the tolerance of LAMP for biological substances was superior than PCR, suggesting the DNA extraction step could be omitted in a LAMP assay. This would save time, labor and cost, which would allow LAMP assay to be developed as a superior diagnostic method for the field test.
Table 1. Effects of biological substances on LAMP and PCR assay