Development of a loop-mediated isothermal amplification assay for the rapid detection of Alongshan virus

Abstract

Alongshan virus (ALSV) is a recently discovered tick-borne zoonotic virus. Currently, there is no rapid and accurate clinical method for ALSV detection. This study aimed to develop a loop-mediated isothermal amplification (LAMP) assay for precise ALSV infection detection. Specific primers were designed based on the S1 segment of the ALSV NE-TH4 strain's genome (GenBank accession no. ON408067.1). The reaction time, temperature and concentration of the neutral red staining solution in the LAMP assay were optimized. Thorough evaluations of specificity, sensitivity and repeatability led to the development of a visually interpretable LAMP assay. The optimal amplification time was 50 min. The minimum detection limit for cDNA was as low as 0.005 pg μl^-1, and sensitivity for standards was 1.68×10³ copies per μl, surpassing that of PCR and real-time PCR. No cross-reactivity was observed with Jingmen tick virus, Bole tick virus 4 and Beiji nairovirus. These results indicate that the LAMP assay is more sensitive and accurate than PCR and real-time PCR. The developed LAMP assay allows for on-site detection, reduces testing costs and provides rapid and accurate results. Thus, it lays a solid foundation for the prevention and control of emerging tick-borne ALSV.

Keywords: Alongshan virus (ALSV); PCR; loop-mediated isothermal amplification technique (LAMP); rapid detection; visualization.

Fig. 1.. Selection and validation of ALSV-LAMP primers. M, DNA Marker DL2000 (TaKaRa, China); N, no template control; P, LAMP assay using cDNA of ALSV genome as template; FP, LAMP assay using non-ALSV cDNA as template. (A) The effectiveness of LAMP primer set selection was evaluated through 1% gel electrophoresis. Three different sets of candidate LAMP primers were subjected to test. (B) The accuracy of the third primer set was confirmed by false-positive verification. This step validated the credibility and practicality of the chosen third primer set, as evident from the results of 1% gel electrophoresis. The reaction mixture comprised 12.5 µl of 2×Lamp Master Mix, 0.8 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 0.16 U µl⁻¹ of DNA Polymerase, 2 µl of template DNA and 5 µl of ddH₂O.

Fig. 2.. Development of neutral red visualization method. M, DNA Marker DL2000 (TaKaRa, China); N, no template control; P, LAMP assay using cDNA of ALSV genome as template. (A) Neutral red visualization LAMP reaction results a. Prior to the initiation of the neutral red LAMP reaction. b. Post the neutral red LAMP reaction. Visual observation of LAMP products accomplished through the pre-addition of neutral red. A noticeable change in colour from orange to red exclusively occurred in tubes containing ALSV genome, while the negative sample retained its orange hue. (B) Authentication of LAMP assay with neutral red pre-addition through electrophoresis. The product generated using the ALSV genome exhibited a distinct amplified band, congruent with the outcome of neutral red colour change. The reaction mixture composition was as follows: 12.5 µl of 2×Lamp Master Mix, 0.8 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 0.16 U µl⁻¹ of DNA Polymerase, 1 µl of neutral red staining solution, 2 µl of template DNA and 4 µl of ddH₂O.

Fig. 3.. Optimization of ALSV-LAMP reaction. M, DNA Marker DL2000 (TaKaRa, China); N, no template control; a. Neutral red visualization of LAMP before reaction. b. Neutral red visualization after LAMP reaction. c. Agarose gel electrophoresis results. (A) Evaluation of different reaction times in ALSV-LAMP experiments. At 65 °C for 60 min, the negative control exhibited no amplified bands. The graph highlights that amplified bands were evident at 65 °C for 50 min. (B) Examination of various reaction temperatures during ALSV-LAMP. At 65 °C for 50 min, the negative control exhibited no amplified bands. Notably, the graph illustrates that amplified bands appeared at 65 °C and after 50 min of reaction. (C) Refinement of the concentration of neutral red staining solution. The most pronounced colour change within the reaction was achieved at a neutral red concentration of 330 µmol l⁻¹. The composition of the reaction mixture was as follows: 12.5 µl of 2×Lamp Master Mix, 0.8 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 0.16 U µl⁻¹ of DNA Polymerase, 1 µl of neutral red staining solution, 2 µl of template DNA and 4 µl of ddH₂O.

Fig. 4.. Specificity assessment of ALSV-LAMP assay. M, DNA Marker DL2000 (TaKaRa, China); N, no template control; a. Prior to the initiation of the neutral red LAMP reaction. b. Post the neutral red LAMP reaction. Lane 1, LAMP products using ALSV genome as template; Lane 2, LAMP products using cDNA extracted from JMTV culture medium as template; Lane 3, LAMP products using cDNA extracted from BLTV4 culture medium as template; Lane 4, LAMP products using cDNA extracted from the tick samples as template. (A, B) Result of colourimetric analysis using neutral red staining solution. The reaction solution with the ALSV template exhibited a change in colour to red. (C) Agarose gel electrophoresis result. The reaction solution with the ALSV template showed an amplified band, consistent with the colour change due to neutral red. The composition of the reaction mixture was as follows: 12.5 µl of 2×Lamp Master Mix, 0.8 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 0.16 U µl⁻¹ of DNA polymerase, 1 µl of neutral red staining solution, 2 µl of template DNA and 4 µl of ddH₂O.

Fig. 5.. Sensitivity analysis of LAMP, PCR and real-time PCR assays. M, DNA Marker DL2000 (TaKaRa, China); N, no template control; a. Neutral red visualization of LAMP before reaction. b. Neutral red visualization after LAMP reaction. c. Agarose gel electrophoresis results. A 10-fold serial dilution (from 1.68×10¹⁰ to 1.68×10³ copies per μl) of the ALSV-LAMP standard was used as a sample for the sensitivity testing. The evaluation of minimum detection limit was executed using serial dilutions of ALSV-cDNA, spanning from 5×10² to 5×10⁻⁴ pg µl⁻¹. (A) Sensitivity testing of the ALSV-LAMP assay. (B) Sensitivity testing of the ALSV-PCR assay. (C) Minimum detection limit of LAMP. (D) Minimum detection limitation of PCR. (E) Sensitivity testing of the ALSV real-time PCR assay. (F) Minimum detection limitation of real-time PCR. The assay detected down to 0.005 pg µl⁻¹ per reaction of ALSV-cDNA for LAMP, and 5 pg µl⁻¹ for PCR and real-time PCR, which showed that the sensitivity of LAMP method was 1,000 times that of PCR and real-time PCR. The composition of the reaction mixture was as follows: 12.5 µl of 2×Lamp Master Mix, 0.8 µM each of FIP and BIP, 0.2 µM each of F3 and B3, 0.16 U µl⁻¹ of DNA polymerase, 2 µl of template DNA and 5 µl of ddH₂O.