In the past several years, numerous multiple-drug-resistant bacteria have been identified: methicillin-resistant Staphylococcus aureus
, the so-called ESKAPE organisms (an acronym for Enterococcus faecium
, S. aureus
, Klebsiella pneumoniae
, Acinetobacter baumannii
, Pseudomonas aeruginosa
, and Enterobacter
species), and others. They are notorious because of their resistance to multiple antimicrobial agents rather than enhanced virulence or pathogenicity. More recently, blaNDM-1
, which is a new mobile class B enzyme or metallo-β-lactamase, has emerged and has added to the antibiotic resistance problem. Bacteria with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are now becoming a major global health problem. The majority of cases have been reported in India (14
) and Pakistan (23
). Cases have also been reported in Australia (25
), Greece (30
), Canada (22
), Singapore (2
), and the United States (1
) and, according to recent reports, in China (36
), Japan (4
), Kenya (26
), Oman (24
), and China's Taiwan region (33
). Development of a sensitive and reliable test for NDM-1 is therefore a priority for early diagnosis and control.
To meet this challenge, we evaluated and optimized a novel LAMP assay for blaNDM-1
detection that was able to specifically detect blaNDM-1
in pathogens that carry blaNDM-1
within 90 min, including DNA extraction. In evaluating the sensitivity of the LAMP assay for blaNDM-1
detection, we observed that the LAMP assay was 100-fold more sensitive than the PCR assay. In addition, the PCR is carried out under temperature-cycling conditions, which is time-consuming, and the reaction also depends on the high precision of the PCR instruments. Compared to PCR, the LAMP reaction was carried out in a constant-temperature environment, and it does not require temperature cycling, so a temperature-controlled water bath or other device that can heat stably is sufficient. Moreover, LAMP reaction primers specifically recognize target sequences of four or six of the six or eight independent target sequence regions, whereas PCR primers recognize target sequences of two independent regions. The specificity and sensitivity are thereby greatly enhanced, and the probability of false-positive results is decreased. Kaneko et al. found that the LAMP reaction is not susceptible to the influence of different components in clinical samples, and so purification of DNA from the sample is not necessary (11
). On the other hand, the sensitivity of the PCR can be greatly reduced in the presence of exogenous DNA and inhibitors. Therefore, the LAMP method is more suitable than PCR for rapid detection of blaNDM-1
in clinical samples. Although the amplification principle of the LAMP method is complex, it is a simple procedure whose rapidity, high sensitivity, and specificity make it suitable for blaNDM-1
detection, especially for routine diagnostic and infection control purposes.
Although there are many advantages of LAMP assay (19
) (LAMP amplifies DNA with high efficiency under isothermal conditions, LAMP is highly specific for the target sequence, and LAMP is simple and easy to perform), it shows a high rate of false-positive results in its current performance. This is because the amplification efficiency of the LAMP assay is extremely high and the LAMP assay is capable of synthesizing 20 μg of specific DNA in a 25-μl reaction mixture within 60 min (15
). Strict spatial separation of reagent preparation and performance of the test is very necessary to avoid contamination. At present, we added low-melting-point paraffin wax to the reaction tubes, after adding reaction solution, to prevent the spread of amplification products. Now it seems that this approach works well for avoidance of contamination.
In conclusion, a specific, sensitive, rapid, and cost-effective LAMP assay for blaNDM-1 detection in pathogens was established. The LAMP assay will be very useful for rapid detection of blaNDM-1 in primary health care units.