A novel high sensitive, specificity duplex enzyme-activated differentiating probes PCR method for the SNP detection and differentiation of MS-H vaccine strains from wild-type Mycoplasma synoviae strains

Abstract

Mycoplasma synoviae (MS) is a contagious pathogen that poses a significant threat to the poultry industry. Detection plays an important role in the prevention and control of MS, particularly in differentiating between wild-type MS and live attenuated vaccine strains for vaccination selection and culling of animals with wild-type only. The live attenuated ts+ vaccine strain MS-H is recognized as the most effective and widely used vaccine. In this study, we have developed a method called double enzyme-activated differentiation probes PCR (DEA-probes PCR) for the differentiation of MS-H vaccine strain from wild-type strain by targeting the single nucleotide polymorphism (SNP) of the 367th nucleotide in the Obg gene sequence. We developed 2 modified probes with the ribonucleotide insert. When the probe perfectly complements with the target, the ribonuclease H2 (RNase H2) will cleave the ribonucleotide, resulting in the generation of fluorescent signal. With a detection limit of 5.8 copies/µL, the DEA-probes PCR method demonstrates 100% specificity in distinguishing wild-type MS from MS-H strains in 1 h. The method demonstrated great performance in real application of 100 superior palate cleft swab samples from chickens in poultry farms. Twenty-eight samples were detected as MS positive, consistent with the results of the Chinese industry standard method. Additionally, our method was able to distinguish 19 wild-type MS strains from 9 MS-H vaccine strains. The DEA-probes PCR method is rapid, specific and sensitive for SNP detection, overcoming the misidentification in MS detection and differentiation. It can be also applied to the differentiation of infected from vaccinated animals (DIVA) for other pathogens.

Keywords: RNase H2 enzyme; mycoplasma synoviae; quantitative PCR detecting; rapid detection; single-nucleotide polymorphism.

Figure 1

Single-nucleotide polymorphism at the 367th site of the Obg gene for the Mycoplasma synoviae serogroup.

Figure 2

Principle of the DEA-probes PCR method and the basic reaction system. (A) The principle of the DEA-probes PCR method established in this study. (B) Basic reaction system with wild-type MS strain standard plasmid and MS-H vaccine strain standard plasmid.

Figure 3

Results of annealing temperature optimization test in 55.0, 55.5, 56.6, 58.2, 60.1, 61.7, 62.6, 63.0. (A) Optimization results for MS-H vaccine strain standard plasmid. (B) Optimization results for wild-type MS strain standard plasmid.

Figure 4

Concentration of differentiating probes test results that the concentration ratio of DEA-probe-MS-H to DEA-probe-MS were 0.25 µM:0.25 µM, 0.25 µM:0.15 µM, 0.25 µM:0.2 µM, 0.25 µM:0.15 µM, 0.25 µM:0.1 µM. (A) Optimization results for MS-H vaccine strain standard plasmid. (B) Optimization results for wild-type MS strain standard plasmid.

Figure 5

Optimization results of MgSO₄ concentration in 5, 6.25, 7.5, and 8.75 μM. (A) Optimization results for MS-H vaccine strain standard plasmid. (B) Optimization results for wild-type MS strain standard plasmid.

Figure 6

Optimization results of RNase H2 enzyme concentration in 0.01, 0.015, 0.02, 0.025, and 0.03 U/µL. (A) Optimization results for MS-H vaccine strain standard plasmid. (B) Optimization results for wild-type MS strain standard plasmid.

Figure 7

Sensitivity test results. (A) DEA-probes PCR method for the detection of plasmid concentrations. MS-H vaccine strain standard plasmid was used at the following concentrations: 5.8 × 10⁴ copies/µL, 5.8 × 10³ copies/µL, 5.8 × 10² copies/µL, 5.8 × 10¹ copies/µL, 5.8 × 10⁰ copies/µL, 5.8 × 10⁻¹ copies/µL. (B) DEA-probes PCR method for the detection of plasmid concentrations. Wild-type MS strain standard plasmid was used at the following concentrations: 5.8 × 10⁴ copies/µL, 5.8 × 10³ copies/µL, 5.8 × 10² copies/µL, 5.8 × 10¹ copies/µL, 5.8 × 10⁰ copies/µL, 5.8 × 10⁻¹ copies/µL.

Figure 8

Specificity test results with wild-type MS strain, MS-H vaccine strain and other 9 pathogen strains.

Figure 9

The process of DEA-probes PCR method in practical application. (A) Sampling with cleft palate swabs. (B) Carrying out the reaction by DEA-probes PCR method. (C) Taking elimination and cleansing or immunization according to the experimental results.