PRV (strain Fa) was obtained from China Institute of Veterinary Drug Control (CIVDC); PRV (strain SA215, Bartha), porcine circovirus-2 (PCV-2), porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), swine transmissible gastroenteritis coronavirus (TGEV), swine vesicular disease virus (SVDV) and swine fever virus (SFV) were all derived from their passage in cell culture which were provided by Shanghai Entry-Exit Inspection and Quarantine Bureau (SHCIQ) (Table 1 ). The clinical samples of porcine brain tissue were collected from suspect cases of pseudorabies.Table 1

Sample list

Total genomic DNA was extracted using the QlAamp^® DNA Blood Mini Kit (Qiagen GmbH, Germany). After elution in 20 μl nuclease-free H₂O, DNA samples were stored at −70 °C until used.

The DNA-binding protein (DBP) gene (GenBank Accession number U80909) was chosen as the suitable target for designing the LAMP primers, as it provides a relatively conserved nucleic acid fragment of 188 bp. Primers were designed using Primer Explorer V3 software (http://primerexplorer.jp/e/).

The basic LAMP reaction was carried out in a volume of 25 μl containing 1× ThermoPol buffer (New England Biolabs Inc., USA), 6.0 mM MgSO₄, 1.0 M betaine (Sigma, Germany), 1.2 mM dNTPs, 0.2 μM each of outer primer, 1.6 μM each of inner primer and 0.4 μM each of loop primer, 8 U of Bst polymerase (Large Fragment; New England Biolabs Inc., USA) with 1 μl total DNA as template. The amplification was performed at 65 °C in a laboratory water bath (Kangle, China, 25–99 °C, ±0.3 °C) for 1 h. Initial experiments were performed to optimize the assay by varying the concentration of MgSO₄, dNTPs, primers, amplification temperature and reaction time.

The amplified products were separated on a 1.5% agarose gel in 0.5× TBE buffer with the voltage of 120 V for 1 h and visualized by staining with ethidium bromide. The result could also be visualized directly with the naked eye according to the white precipitate of magnesium pyrophosphate generated in the reaction or the green color produced by the intercalating dye Picogreen^® (Invitrogen, USA). To confirm the specificity of amplification products, 2 μl of the reaction mixture was digested with Hinc II at 37 °C for 4 h.

The sensitivity of LAMP was demonstrated and compared with PCR (Balasch et al., 1998, Chen et al., 2002, Lee et al., 2007) using various PRV DNA dilutions (10⁻¹ to 10⁻⁹) as templates (original concentration of DNA sample was 10 ng/μl).

The specificity of LAMP was examined by the use of DNA (or cDNA) extracted from three different PRV strains and six other viruses known to be related genetically to PRV or to cause similar clinical signals in pig.

The evaluation of the LAMP assay was carried out using DNA extracted from clinical samples with PCR running in parallel.

LAMP primers were designed using the Primer Explorer V3 software based on a conserved fragment of the DBP gene (Fig. 1 ). After screening according to the end stability of the primers (the free energy of the 3′ ends of F2/B2, F3/B3, LF/LB and the 5′-end of F1c/B1c should be −4 kcal/mol or less), one set of primers was chosen (Table 2 ). The outer primers (F3 and B3) help to displace the primary strand. The inner primers (FIP and BIP) each have two distinct sequences corresponding to the sense and anti-sense sequence of the target. FIP comprises F1c and F2 while BIP comprises B1c and B2. The forward loop primer (LF) is designed using the complementary strand corresponding to the region between F1 and F2 while reverse loop primer (LB) was designed using the complementary strand corresponding to the region between B1 and B2.Fig. 1

The conserved fragment of the DBP gene used to design LAMP primers (A) and the schematic diagram of LAMP primers (B).

The LAMP reaction conditions were optimized by varying the concentration of MgSO₄, dNTPs, primers, amplification temperature and reaction time. The results indicated that the reaction could be carried out when the Mg²⁺ concentration is higher than 4 mM and the optimal amplification was got at 8 mM (Fig. 2(A)). As shown in Fig. 2(B), the ladder-like bands could be obtained with dNTPs ranging from 0.4 to 1.2 mM while 1.0 mM is the best. Fig. 2(C) demonstrates that the target gene was amplified in the primer ratios ranging from 1:1 to 1:10 while the ratio at 1:8 gave the best amplification. As shown in Fig. 2(D and E), the amplification could be detected initially at 0.5 h and reached maximal at 1 h while the most clear pattern could be obtained at 63 °C.Fig. 2

Optimization of the LAMP reaction for PRV detection. (A) Effect of Mg²⁺ on the LAMP reaction: 1–6, Mg²⁺ was 2, 4, 6, 8, 10 and 12 mM, respectively. (B) Effect of dNTPs on the LAMP reaction: 1–7, dNTPs was 0, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mM, respectively. (C) The effect of ratio of outer and inner primers on the LAMP reaction: 1–5, ratio was 1:1, 1:2, 1:4, 1:8 and 1:10, respectively. (D) The effect of temperature on the LAMP reaction: 1–3, temperature located at 60, 63 and 65 °C, respectively. (E) The effect of reaction time on the LAMP reaction: 1–4, the amplification time was 15, 30, 45 and 60 min, respectively.

Amplification products of LAMP were detected by agarose gel electrophoresis and visual inspection. As shown in Fig. 3(A), the target DNA sequence was amplified at 63 °C with a ladder-like pattern on the gel. The positive reaction was also indicated clearly by the white precipitate of magnesium pyrophosphate in Fig. 3(B) and the green color produced by the intercalating dye Picogreen^® shown in Fig. 3(C). The specificity of the amplification product was confirmed by Hinc II digestion as indicated in Fig. 3(A, lane 2). Products of predictable sizes of the 80 and 108-bp motifs were resolved on the gel.Fig. 3

Agarose gel electrophoresis (A) and visual inspection of LAMP (B and C). 1, LAMP products of PRV; 2, LAMP products digested with Hinc II; 3, negative control; 4, positive LAMP reaction visualized by turbidity; 4′, negative LAMP reaction visualized by turbidity; 5, positive LAMP reaction visualized by adding Picogreen^®; 5′, negative LAMP reaction with Picogreen^®.

The sensitivity of LAMP was demonstrated and compared with PCR tests by using various PRV DNA dilutions (10⁻¹ to 10⁻⁹) as templates. As shown in Fig. 4 , LAMP was able to detect template at 10⁻⁶ dilution (about 10 fg DNA), whereas PCR could only amplify the 10⁻³ to 10⁻⁴ dilution DNA sample. Therefore, the detection sensitivity of LAMP was 100–1000-fold higher than the PCR.Fig. 4

Sensitivity of LAMP for PRV detection. (A) The result obtained by LAMP. (B–D) The results obtained according to PCR tests by Chen et al. (2002), Balasch et al. (1998) and Lee et al. (2007), respectively. 1–9, DNA sample located at 10⁻¹, 10⁻², …, 10⁻⁹ dilutions, respectively; 10, negative control.

Three different PRV strains and six other viruses were investigated to confirm the specificity of the LAMP for PRV detection. The results showed that only the PRV is amplified while no amplification is performed in all other tested viruses (Fig. 5 ).Fig. 5

Specificity of LAMP for PRV detection. 1–9, PRV strain Fa, SA215, Bartha, porcine circovirus-2, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, swine transmissible gastroenteritis coronavirus, swine vesicular disease virus and swine fever virus, respectively; 10, negative control.

The evaluation of the LAMP assay was carried out using DNA extracted from five clinical samples with PCR running in parallel. The results of LAMP assay correlated well with the PCR, meanwhile, sample 2 was positive by LMAP, but not amplified by PCR (Table 3 ).Table 3

The LAMP assay and PCR with clinical samples