Internal heating method of loop-mediated isothermal amplification for detection of HPV-6 DNA

Abstract

Loop-mediated isothermal amplification (LAMP) is a promising diagnostic tool for genetic amplification, which is known for its rapid process, simple operation, high amplification efficiency, and excellent sensitivity. However, most of the existing heating methods are external for completion of molecular amplification with possibility of contamination of specimens. The present research provided an internal heating method for LAMP using magnetic nanoparticles (MNPs), which is called nano-LAMP. Near-infrared light with an excitation wavelength of 808 nm was employed as the heating source; hydroxy naphthol blue (HNB) was used as an indicator to conduct methodological research. We demonstrate that the best temperature was controlled at a working power of 2 W and 4.8 µg/µL concentration of nanoparticles. The lowest limit for the detection of HPV by the nano-LAMP method is 10² copies/mL, which was confirmed by a gel electrophoresis assay. In the feasibility investigation of validated clinical samples, all 10 positive HPV-6 specimens amplified by nano-LAMP were consistent with conventional LAMP methods. Therefore, the nano-LAMP detection method using internal heating of MNPs may bring a new vision to the exploration of thermostatic detection in the future.

Keywords: Genetic amplification; Human papillomavirus; Loop-mediated isothermal amplification; Magnetic nanoparticles; Nano-LAMP.

Scheme 1

A depicts the synthesis process of magnetic nanoparticles (MNPs) with negative charges. B illustrates of the nano-LAMP experimental process

Fig. 1

A SEM of MNPs. (B) ζ potential of MNPs, HPV DNA and MNPs + DNA. (C) The concentration of the product and concentrations of the supernatant and the surface of MNPs was measured after three washes and re-suspension was measured by the NanoDrop-2000

Fig. 2

A The NIR thermal images of nano-LAMP mixture recorded by infrared camera during a 808-nm laser irradiation. (B) NIR thermal images of droplets containing MNPs at 40 μg/μL, and ultrapure water droplet with laser off (B) and laser on (C). (D) Thermal imager recording of 25 min of temperature change. (E) Photothermal conversion curves of different concentrations (n = 3) and different powers (F) (n = 3). (G) Thermal images of different samples recorded by infrared camera during 808-nm laser irradiation. (H) Photothermal transformation curve of five cycles

Fig. 3

A Images of pre-reactions for conventional LAMP (conventional LAMP + and conventional LAMP −) and reactions added MNPs. (B) Post-reaction images for nano-LAMP (nano-LAMP + and nano-LAMP −) after magnetic separation. (C) Gel electrophoresis images of LAMP products in A and B. (D) Color contrast pictures and electrophoretic contrast of HPV DNA diluted to different concentrations (10⁷ copies/mL–10¹ copies /mL) of HPV-6 DNA were involved in conventional LAMP reactions and in nano-LAMP reactions (E)

Fig. 4

Results of cross-reaction experiment: primers of HPV-6 and different types of DNA (HPV-6, HPV-42, HPV-43, HPV-44). (A) Images of pre-reactions for conventional LAMP, reactions added MNPs and post-reaction images for nano-LAMP after magnetic separation. (B) Gel electrophoresis images of nano-LAMP products

Fig. 5

Clinical sample feasibility verification. Ten positive samples of HPV-6 were randomly selected for nano-LAMP and conventional LAMP. (A) Images of pre-reactions for conventional LAMP reactions added MNPs and post-reaction images and for nano-LAMP after magnetic separation (B). (C) Gel electrophoresis images of conventional LAMP products and gel electrophoresis images of nano-LAMP products (D). (E) Results of cross-reaction validation of clinical samples: Primers of HPV-6 were reacted with clinical samples (HPV-6, HPV-11, HPV-16, and HPV-42–44) for conventional LAMP (F) and nano-LAMP (G)