A primer-probe set for differentiating the vaccine and field strains of peste des petits ruminants (PPR) virus

Abstract

The invention relates to a primer-probe set, comprising a forward primer having the nucleotide sequence of SEQ ID NO:1 and a reverse primer having the nucleotide sequence of SEQ ID NO:2, and a peptide nucleic acid (PNA) probe having the sequence of HEX-OO-(SEQ ID NO:3)-Lysine-BHQ1, which are used for differentiating the Peste des Petits Ruminants (PPR) virus vaccine and field strains and for preventing false positive results arising from the PPR vaccine, as well as to their operating temperatures. Said primer-probe set can differentiate between the vaccine strain containing the nucleotide sequence of SEQ ID NO:4 and the field strain containing the nucleotide sequence of SEQ ID NO:5. Thus, by means of said primer-probe set, false positive results arising from the PPR vaccine are prevented, and accordingly, unnecessary quarantine measures are also avoided.

Description

[0001] A PRIMER-PROBE SET FOR DIFFERENTIATING THE VACCINE AND FIELD STRAINS OF PESTE DES PETITS RUMINANTS (PPR) VIRUS

[0002] Technical Field

[0003] The invention relates to a primer-probe set developed for differentiating the vaccine and field strains of the Peste des Petits Ruminants (PPR) virus. The probe in the primer-probe set is used in a real-time polymerase chain reaction (RT-qPCR) technique operating based on the principle of a probe-based fluorescence melting curve analysis (FMCA) and by means of this primer-probe set, false positives arising from the PPR vaccine strain are prevented.

[0004] State of the Art

[0005] Peste des Petits Ruminants (PPR), also known as sheep and goat plague, is a contagious viral disease frequently observed in many parts of the world, including our country. PPR is not a zoonotic disease and does not spread to humans; however, it causes numerous health problems in small ruminants. PPR may cause symptoms such as high fever, diarrhoea, and coughing, and may also result in death. Since the rate of transmission of the disease is extremely high, various quarantine measures are taken after its detection. Therefore, the first step in combating PPR is the early detection of the disease and the implementation of other necessary measures (such as quarantine and vaccination). Currently, competitive ELISA and PCR techniques are widely used as laboratory tests for the diagnosis of PPR. Once PPR is diagnosed by any of these techniques, the necessary quarantine measures are implemented, and treatment of the infected animals is initiated. In addition to the implementation of quarantine measures to prevent the spread of the disease, other protective and control measures such as disinfection and vaccination are also applied. As in many viral diseases, vaccines have been developed for PPR, and these vaccines play an important role in protecting animals against the PPR virus.

[0006] PPR vaccines are used in many regions of the world to protect sheep and goats against the PPR virus. The PPR vaccine contains an attenuated PPR virus with reduced pathogenicity. However, this attenuated PPR vaccine virus may cause false positive results in PCR tests, which are widely used for the diagnosis of the disease. The PCR test used in routine diagnosis cannot distinguish between the attenuated PPR vaccine strain present in the vaccine and the actual pathogenic field strains found in samples suspected of infection. This leads to false positive results and consequently to unnecessary vaccination and quarantine practices aimed at controlling the disease. Unnecessary quarantine measures cause loss of time and economic losses due to unnecessary vaccination and the prevention of trade.

[0007] In the state of the art, a study conducted by Oz et al. aimed to differentiate PPR vaccine and field strains [1], In said study, the differentiation of PPR vaccine and field strains was attempted using PCR analysis with EvaGreen dye. Although said technique is more economical compared to PCR techniques using probes, it requires extreme care in terms of optimisation. This is because EvaGreen dye binds to all double-stranded DNA fragments present in the medium and may lead to undesired positive results. Even a minor problem during optimisation may lead to such undesired bindings and consequently to false positive results. Moreover, since each DNA strand to which EvaGreen dye binds carries a negative electrical charge, a repulsive electrical force occurs as a result of the tendency of similar charges to repel each other. As this repulsive force contributes to the separation of DNA strands, it adversely affects melting curve analyses. For this reason, in said study, the temperature difference between the vaccine and field strains in the melting curve analysis was as low as 0.5°C, which led to the misinterpretation of the results. Although the method described in said study is advantageous in terms of cost, it involves many factors that may result in false positive results.

[0008] In another study in the state of the art, Lamien et al. used the fluorescence resonance energy transfer probe (FRET) technique to differentiate between sheep poxvirus (SPPV) and goat poxvirus (GTPV), which cause sheep and goat pox disease in small ruminants, and lumpy skin disease virus (LSD), which causes lumpy skin disease in cattle [2], Said technique is a primer-probe-based technique that requires the use of multiple primer-probe pairs. This constitutes a disadvantage for optimisation compared to the use of a single primer-probe pair. Due to the chemical structure of the probes used, the salt concentration of the PCR mixture is another important parameter in terms of optimisation. Furthermore, the issue of negative electrical charge, described in detail in the study by Oz et al., also applies to this study. Considering all these factors together, the proper operation of the technique requires greater effort, time, and cost. In addition, the increased optimisation risks raise the likelihood of the technique failing to function and producing false positive results.

[0009] The limitations and inadequacies of the existing solutions in the state of the art, the high probability of obtaining false positive results due to errors occurring during the optimisation of the techniques used for differentiating PPR vaccine and field strains, the resulting loss of time and money during optimisation processes, the need to design separate primer-probe pairs for each virus (as in the study by Lamien et al.), and most importantly, the absence of a primer-probe pair available for PPR that could be used for the intended purpose of the present study, have together created the need for a development in this field.

[0010] Brief Description and Aims of the Invention

[0011] The invention describes a primer-probe set used for differentiating the vaccine and field strains of the Peste des Petits Ruminants (PPR) virus. By means of this method, false positive results arising from the PPR vaccine in real-time polymerase chain reaction (RT-qPCR) tests are prevented. Thus, both the implementation of unnecessary quarantine measures and the associated economic losses are avoided.

[0012] The main aim of the invention is to prevent false positive PPR results arising due to vaccination. The primer-probe set that is the subject of the invention can differentiate between PPR vaccine and field strains and prevents financial losses caused by unnecessary quarantine measures resulting from false positive results associated with the vaccine strain. Furthermore, by means of the primer-probe set that is the subject of the invention, sequencing analyses required for this differentiation process are no longer needed. In this way, savings are achieved in terms of both labour and expenditures required for sequencing analysis.

[0013] Another aim of the invention is to eliminate uncertainties arising from weakly positive PPR results of suspicious nature. As mentioned above, the differentiation process is carried out by means of sequencing analyses. However, strong positive results are required in order to perform sequencing analysis. Unfortunately, weak positive results often cannot be transferred for sequencing analysis. By means of the primer-probe set that is the subject of the invention, weak positive results that are too low to be transferred to sequencing analysis can be tested, thereby clarifying cases of suspected positivity. PPR is a notifiable disease, and the World Organization for Animal Health aims to achieve its complete eradication worldwide by 2030. Falsely detected positive results hinder international animal trade and adversely affect eradication efforts.

[0014] Description of Drawings

[0015] Figure 1 : Alignment of the primer-probe set with the target gene with a) forward primer; b) SEQ ID NO: 3; c) reverse primer. (Dots (.) indicate the match between the primerprobe set and the target genes, while letters (A, C, T) indicate mismatches (mutation(s)).)

[0016] Figure 2: Melting points obtained as a result of fluorescence melting curve analysis. (Dashed line: vaccine sample (vaccine strain): 65°C melting point; dotted line: field sample (field strain): 48°C melting point; solid line: negative control.)

[0017] Detailed Description of the Invention

[0018] The invention relates to a primer-probe set comprising a forward primer having the nucleotide sequence of SEQ ID NO:1 , a reverse primer having the nucleotide sequence of SEQ ID NO:2, and a probe having a peptide nucleic acid (PNA) structure represented by HEX-OO-(SEQ ID NO:3)-Lysine-BHQ1 , which is labelled with 6- hexachloro-fluorescein (HEX) and black hole quencher 1 (BHQ1 ) dyes, used for differentiating the Peste des Petits Ruminants (PPR) vaccine and field strains and for preventing false positive results arising from the PPR vaccine. By means of said primer-probe set, false positive results caused by the PPR vaccine strain containing the nucleotide sequence of SEQ ID NO:4 and the field strain containing the nucleotide sequence of SEQ ID NO:5, which appear in routine diagnostic RT-PCR and RT-qPCR tests, are prevented through the real-time polymerase chain reaction (RT-qPCR) method operating based on the principle of a probe-based fluorescence melting curve analysis (FMCA). Thus, both the implementation of unnecessary quarantine measures and the associated economic losses are prevented.

[0019] The primers (SEQ ID NO:1 and SEQ ID NO:2) in the primer-probe set that is the subject of the invention have the same chemical structure as the target gene (DNA). However, their nucleotide sequences are specific to the PPR virus and have been designed exclusively for this invention. The probe having the nucleotide sequence of HEX-OO-(SEQ ID NO:3)-Lysine-BHQ1 possesses a peptide nucleic acid (PNA) structure and is one of the fundamental elements of the invention. The probe is chemically structured as a peptide nucleic acid (PNA) comprising N-(2-aminoethyl)- glycine units and peptide bonds, with a backbone carrying a NEUTRAL electrical charge. The target gene (DNA) consists of deoxyribose sugar and phosphate groups and has a negative electrical charge due to the phosphate bonds it contains. Due to its chemical structure, PNA binds to the negatively charged target gene (DNA) more tightly and firmly, without any repulsive electrical force. In addition, due to its chemical composition, it is not degraded by the enzymes used in the PCR mixture and is not adversely affected by the salt concentration of the mixture (master mix). This enables the test to yield more specific and reliable results and ensures a greater temperature difference during strain separation in melting curve analysis, allowing the results to be clearer and more precise. For these reasons, it provides an advantage over the studies included in the existing technique (Oz et al. and Lamien et al.). In Figure 1 , the alignment of the forward primer having the nucleotide sequence of SEQ ID NO:1 , the reverse primer having the nucleotide sequence of SEQ ID NO:2, and the PNA probe sequence containing SEQ ID NO:3 with the gene regions targeted in the PPR vaccine strain containing SEQ ID NO:4 and the field strain containing SEQ ID NO:5 is shown. In Figure 1 , black dots represent complete alignment, whereas letters (A, C, T) indicate mutations. Said probe is labelled with HEX and BHQ1 dyes for fluorescence emission, and -00- rings and Lysine (AAG / AAA) are added to increase solubility, being designed as HEX-OO-(SEQ ID N0:3)-Lysine-BHQ1 . Mutation points are designed to be located at the centre of the probe in order to create a melting temperature (Tm) difference of at least 5°C or higher. The genome of the PPR vaccine strain is 1578 nucleotides in length. In the invention, the region containing the nucleotide sequence of SEQ ID N0:4 in the vaccine strain and the region containing the nucleotide sequence of SEQ ID N0:5 in the field strain are targeted. In Figure 1 , the sequence between 425-605 nt of SEQ ID NO:4 and SEQ ID NO:5 is presented. In Figure 1 , the regions targeted by the primer-probe set that is the subject of the invention are shown; untargeted regions are not indicated in Figure 1 (the sequences between 450-500 nt and 520-580 nt are not shown in Figure 1 ). As can be seen in Figure 1 , in the region containing the sequence of SEQ ID NO:4 of the vaccine strain, the positioning (forward primer 426-448 nt, probe 502-516 nt, reverse primer 583-605 nt) and alignment of the primer-probe set in the target gene region are shown. Furthermore, in Figure 1 , dots (.) indicate the alignment between the primer-probe set and the target genes, whereas letters (A, C, T) indicate mismatches (mutations). The probe shows a complete match with the vaccine strain, while, as planned, it shows mismatches with the field strain containing the nucleotide sequence of SEQ ID NO:5 at the 510th and 511th nucleotides (A and C, respectively).

[0020] In order to determine the optimal annealing temperature of the primers (SEQ ID NO:1 and SEQ ID NO:2), a gradient conventional RT-PCR analysis (simultaneous reactions at different annealing temperatures) was performed, and it was determined that they annealed at an average temperature of 53.8°C. Subsequently, to optimise RT-qPCR analysis, PCR mixtures were prepared under asymmetric and non-asymmetric PCR conditions and with different probe concentrations. For this purpose, in a total reaction volume of 20 pl, a PCR mixture was prepared containing final concentrations of 250 nMol forward primer (SEQ ID NO:1 ), 500 nMol reverse primer (SEQ ID NO:2), and 125 nMol probe (HEX-OO-(SEQ ID NO:3)-Lysine-BHQ1 ), in which the melting curve analysis of both strains showed clearer differentiation (Figure 2). At concentrations other than these, a decrease in PCR efficiency was observed.

[0021] Table 1 : RT-qPCR Mixture Table 2: RT-qPCR Temperature Conditions

[0022] In order to test the specificity of the primers (SEQ ID NO: 1 and SEQ ID NO:2) and the probe (HEX-OO-(SEQ ID NO:3)-Lysine-BHQ1 ), in addition to the PPR vaccine (SEQ ID NO:4) and field samples, Pestivirus, Rotavirus, Coronavirus, and Distemper virus, which are routinely used as positive controls in standard tests, were tested in triplicate by means of RT-qPCR. Accordingly, it was determined that the PPR strains produced a positive amplification curve and a melting curve, whereas the others did not. The fact that the designed primer-probe set did not exhibit any cross-reaction with viruses from the same family as the PPR virus, such as the Distemper virus, or with viruses from different families, such as Pestivirus, Rotavirus, and Coronavirus, which may be present in suspected samples during routine diagnosis, and reacted only with the PPR vaccine and field strains, demonstrates that the designed primer-probe set is specific to the target virus and does not interact with other viruses, thereby eliminating the possibility of false positive results. This constitutes one of the most important criteria of the method. Thus, it has been determined that the primer-probe set that is the subject of the invention is specific (unique) to the PPR virus and does not interact with any other viruses. REFERENCES

[0023] [1] Oz, ME., Torlak, E., 2022. A high-resolution melting assay to differentiate a peste des petits ruminants virus vaccine strain from field isolates in Turkey. Arch. Virol. 167.

[0024] [2] Lamien, C. E., Lelenta, M., Goger, W., Silber, R., Tuppurainen, E., Matijevic, M., Luckins A. G., Diallo A., 2011. Real time PCR method for simultaneous detection, quantitation and differentiation of capripoxviruses. Journal of virological methods, 171 (1 ), 134-140.

Claims

CLAIMS1. A primer-probe set developed for differentiating the vaccine strain and the field strain of the Peste des Petits Ruminants (PPR) virus, comprising:- a forward primer having the nucleotide sequence of SEQ ID NO:1 ,- a reverse primer having the nucleotide sequence of SEQ ID NO:2, and- a probe having a peptide nucleic acid (PNA) structure represented by HEX- OO-(SEQ ID NO:3)-Lysine-BHQ1 , which is labelled with 6-hexachloro- fluorescein (HEX) and black hole quencher 1 (BHQ1 ) dyes.

2. A primer-probe set according to claim 1 , wherein said PPR virus vaccine strain comprises the sequence of SEQ ID NO:4, and said field strain comprises the sequence of SEQ ID NO:5.

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