Development of loop-mediated isothermal amplification assay for specific and rapid detection of differential goat Pox virus and Sheep Pox virus

Sheep pox and goat pox are economically important diseases in goat and sheep producing areas of the world. Sheep pox and goat pox result from infection by SPPV or GTPV respectively, and are closely related members of the Capripoxvirus genus in the family Poxviridae. Clinically, sheep pox and goat pox have indistinguishable symptoms. Several PCR-based assays have been reported to distinguish SPPV from GTPV including cleaved amplification polymorphism sequence-tagged sites (RFLP-PCR) and real-time PCR (qPCR) [1–5]. In our laboratory, distinction of GTPV from SPPV was established via a Hinf I digest of the p32 gene, followed by a sequence alinment of G-protein-coupled chemokine receptor (GpCR) genes [5, 6]. Some of the advantages of qPCR include speed, sensitivity, and real time monitoring to determine exact concentrations. However, this approach requires expensive high precision instrumentation and specialized training for operation and data analysis, presenting a need for a more convenient alternative that is robust, inexpensive, and easy to operate and maintain.

Recently, loop-mediated isothermal amplification (LAMP) has been developed for the diagnosis of a number of diseases [1, 7, 8]. The LAMP reaction can be conducted under isothermal conditions ranging 60-65°C by using four or six primers recognizing six or eight distinct regions [9]. LAMP produces large quantities of amplified product resulting in easy visual detection either via turbidity or fluorescence [10]. The present study established the ability of LAMP assays to differential detect GTPV and SPPV through the targeting of inverted terminal repeat (ITR) sequences. Compared to conventional PCR techniques, the newly established LAMP assay is simple, efficient, cost-effective and convenient, making it a useful diagnostic tool for clinical samples.

GTPV and SPPV contain double-stranded DNA genomes that are approximately 150 kbp and share at least 147 putative genes, to include conserved poxvirus replicative and structural genes and genes likely involved in virulence and host range [11]. Restriction endonuclease analysis and cross-hybridization studies of SPPV and GTPV indicate that these viruses, although closely related (estimated 96 to 97% nucleotide identity), can be distinguished from one another and may undergo recombination in nature [11–14]. Several PCR tests have been developed for the detection of Capripoxviruses[5, 6, 15–24]. In our laboratory, distinction of GTPV from SPPV was established via a Hinf I digest of the p32 gene, followed by sequence alinment of GpCR genes [5, 6]. However, these methodologies are time consuming, expensive and require experienced laboratory staff. This presents a real need for a more convenient alternative to PCR that is robust, inexpensive, and easy to operate and maintain.

LAMP is a novel nucleic acid isothermal amplification technique developed by Notomi [25] and serves as a powerful gene amplification tool due to its high specificity and sensitivity under isothermal condition [26–28]. Previous LAMP methods were developed for the rapid detection of Capripoxviruses[1] and were unable to distinguish SPPV from GTPV. The present study aimed to develop a LAMP method for the rapid distinction of SPPV from GTPV, and to evaluate its applicability through field sample testing. LAMP primer design was based on six regions in the target sequence designated from the 5’-end as F3, F2, F1, B1, B2, and B3 (Figure 1). The forward inner primer (FIP) consists of the F2 sequence (at its 3’ end) that is complementary to the F2c region and the same sequence as the F1c region at its 5’ end. The four key factors in the LAMP primer design are the T_m, primer end stability, GC content and secondary structure. T_m is estimated using the Nearest-Neighbor method, which is an approximation method that provides values closest to the actual values. The T_m for each region was determined to be ~ 65°C (64 - 66°C) for F1c and B1c, ~ 60°C (59 - 61°C) for F2, B2, F3, and B3 and ~ 60°C for the loop primers. Since primers serves as the starting point of DNA synthesis, a certain degree of stability must be achieved. The 3’ ends of F2/B2, F3/B3, and LF/LB and the 5’ end of F1c/B1c were designed to have a free energy of -4 kcal/ mol or less. The 5’ end of F1c after amplification corresponds to the 3’ end of F1, making its stability important. Primers were designed to have GC content between ~40% to 65%, with 50% to 60% GC content optimal. It is important, particularly for inner primers, that primers are designed to eliminate the formation of secondary structures. Additionally, it is important to prevent primer dimerization by ensuring that the 3’ ends are not complementary.

The most critical aspect of the current study is to design robust primers able to achieve differential detection of SPPV and GTPV, thus warranting a rigorous design process. To optimize SPPV primer specificity, primers comparison analysis shows that B2 of the SPPV primers (that is the composition of SPPV BIP) is a characteristic sequence in the SPPV genome (see SPPV SFIP underlined sequence in Table 1) and does not exist in the GTPV genome. The calculated dimer (minimum) dG SPPV LAMP primer was -2.49 kcal/mol, the 3’ ends of F2/B2 and F3/B3 and the 5’ end of F1c/B1c were designed to have a free energy of -4 kcal/mol or less and GC rates were around 0.4. The calculated T_m for F3 was 55.67 and for B3 was 55.34, making both of the T_ms relatively close. Additionally, the calculated T_m for F1c was 60.08 and for B1c was 60.10, yielding very close T_ms for both. Lastly, the calculated T_m for F2 was 56.96 and for B2 was 55.42, again yielding close T_ms. These indicators are more in line with the general LAMP primer design requirements generating high specificity and sensitivity in theory, and which was experimentally validated. The SPPV primer achieved high sensitivity and specificity in the presence of 1.037 × 10⁴ copies of DNA template.

In order to guarantee GTPV primer specificity, primer sequence specificity was assessed via comparative analysis to shows that B3/F2 of the GTPV primers (that is, the composition of GTPV FIP) and B2 of the GTPV primers (that is the composition of GTPV BIP) are characteristic sequences in the GTPV genome (see GTPV GF3, GFIP and GBIP underlined sequences in Table 1) and does not exist in SPPV genome. The designed GTPV LAMP primers had higher specificity than SPPV primers in theory due to a GC rate around 0.4, the 3’ ends of F2/B2 and F3/B3 and the 5’ end of B1c were designed with a free energy of -4 kcal/mol. The calculated T_m for F3 was 56.01 and for B3 was 56.13, making them relatively close. Additionally, the calculated T_m for F1c was 61.20 and for B1c was 60.03, also making them relatively close. Lastly, the calculated T_m for F2 was 56.21 and for B2 was 56.39, yielding very close T_ms. These indicators are more in line with the general LAMP primer design requirements. The calculated dimer (minimum) dG GTPV lamp primer was only -1.18 kcal/mol, and 5’ end of F1c had a free energy of only -3.90 kcal/mol, less than the target -4 kcal/mol. These parameters should results in primers with higher specificity but lower sensitivity in theory, which was experimentally validated. The specificity of the SPPV primer was high, but the sensitivity was lower as demonstrated by a need of 1.045 × 10⁶ copies of template.

All GSPV primers were designed to match all sequences characteristics in the GTPV and SPPV genomes, with indicators more in line with general LAMP primer design requirements. The only drawback in the design was the 5’ end of F1c having a free energy of -3.48 kcal/mol, which was less than the ideal -4 kcal/mol, but the predicted high degree of specificity and adequate sensitivity were experimentally validated. The specificity of the GSPV primers was high, in addition to achieving a high sensitivity as demonstrated by the use of 1.037 × 10³ copies of template.

Although the predicted SPPV primer specificity could have been higher, experimentation showed its inability to produce an amplification product from other pathogenic genomes, thus confirming the ability of the SPPV primer to specifically detect SPPV alone. While both predictive and experimental evidence displayed a high degree of specificity in GTPV primers, but a lower sensitivity, which can be rectified through its combining with the GSPV primers. In short, combining all three sets of primers enables the quick and efficient detection of GTPV and SPPV. While the methods established in this study are effective, they could be further optimized by designing loop primers to further reduce experimentation time and visualization could be more streamlined through the utilization of fluorescence dyes.

In clinical samples testing found that SPPV LAMP primer detected miss one case which can be detected by GSPV LAMP primer. The possible reason is that all SPPV nucleic acid concentration in the samples were within the scope of SPPV detection sensitivity, but the nucleic acid content of the miss sample was smaller than the SPPV LAMP highest sensitivity(1.037 × 10⁴ copies) but within GSPV LAMP primer detection sensitivity,(only reach to 1.037 × 10³ copies). So the judgment of the samples should be to test again, or in other ways for further confirmation, and pay more attention to the concentration of the sample in the process of sample handling.

The presented experimentation has shown that the sequence of the GTPV primer can provide specificity and rapid detection of GTPV nucleic acids, but was unable to detect SPPV nucleic acids under the same conditions. On the other hand, the SPPV primer can provide specificity and rapid detection of SPPV nucleic acids, but was unable to detect GTPV nucleic acids under the same conditions. However, the GSPV primer can rapidly amplify GTPV nucleic acid and SPPV nucleic acid. Collectively, GSPV, GTPV and SPPV LAMP primers when combined possess the analytical ability to fully distinguish between GTPV and SPPV.

These laboratory studies showed that the LAMP method of differential GTPV and SPPV detection is inexpensive, rapid, simplistic, specific, and sensitive. These attributes make this method an optimal detection system for field detection and differential diagnosis of GTPV and SPPV. It is a promising assay for extensive application and rapid diagnosis of GTPV and SPPV infection in the laboratory and the field, especially in countries that lack the resources needed for molecular diagnostic techniques.