KEYWORDS
Peste des Petits Ruminants, real-time qRT-PCR, melting curve analyses,
peste des petits ruminants virus types Ⅱ and Ⅳ
Peste des Petits Ruminants (PPR) is a highly contagious, cross-species
disease caused by the Peste des Petits Ruminants Virus (PPRV) (Abubakar
et al. 2017; Njeumi et al. 2020). It can infect not only small ruminants
such as goats and sheep, but also large ruminants such as camels,
buffalo, and wild animals (Lembo et al. 2013; Zakian et al. 2016).
According to 2018 data from the FAO, there are 2.5 billion small
ruminants and over 68% of sheep and goats living in PPR-infected
countries (Zhao et al. 2021). The widespread distribution and prevalence
of PPR have an important impact on economic trade and biodiversity.
PPRV belongs to the Morbillivirus genus, Paramyxoviridaefamily (Gibbs et al. 1979). According to the sequences of the N gene and
F gene, PPRV can be classified into four lineages, I, II, III, and IV
(Balamurugan et al. 2010). Lineage I and II are predominantly endemic in
West Africa (Muniraju et al. 2014b; Tounkara et al. 2018), while lineage
III is mainly found in the Arab region, and has also been reported in
East Africa (Muniraju et al. 2014a; Rume et al. 2019), Lineage IV is
mainly prevalent in the Middle East and Asia, and has also been reported
in Africa (Woma et al. 2016; Liu et al. 2018; Tounkara et al. 2018). PPR
was first reported in the Tibet region in 2007 (Wang et al. 2009), then
re-emerged in Xinjiang Uyghur Autonomous Region in 2013, and then spread
to 23 provinces, autonomous regions, and municipalities of China (Bao et
al. 2017; Li J. et al. 2017). China has adopted strict compulsory
immunization and culling policies, which have effectively controlled the
occurrence of the PPR. Currently, the live vaccine named the Nigeria
75/1 is the only vaccine widely used for the prevention of PPR in China,
it can provide strong immune protection in goats and sheep, as it can
proliferate continuously after injection. In clinical application, as an
inability to distinguish vaccine group from PPRV antibody or antigen
positive animals is the significant impediment to the eradication of
PPR. So, for prevention and eliminating PPR, there is a great need for a
simple assay that can detect and distinguish the PPRV vaccinated and
infected group at the same time.
Since the Nigeria 75/1 strain PPRV belongs to lineage II, while the PPRV
prevalent in China all belong to type IV (Bao et al. 2017), an SYBR
Green I real-time RT-qPCR assay was developed by analyzing all the
published PPRV sequences of type II and type IV. We use only one pair of
primers that can detect all the type II and IV PPRV, and by analyzing
different melting curve analyses (MCA), it can differentiate them
conveniently and quickly. The parameters were optimized, and the
sensitivity, specificity, and reproducibility of the assay were assessed
and compared with the traditional methods. The diagnostic application of
the assay was carefully evaluated based on the PPRV available sequences.
First, 71 published whole-genome sequences (9 form type II PPRV and 62
form type IV PPRV, including 37 were Chinese strains and 25 were foreign
strains) were retrieved from the GenBank database. DNAstar software was
used for comparison, and L gene was used to design primers to detect and
differentiate type II and IV PPRV, based on the principle that GC
content difference in the inner region of two typed PPRV gene sequences
can produce different melting curves. The optimized forward primer F was
5’-ACAGGTTCGACAACATTCAAGCCA-3’ and
reverse primer R was
5’-GCGAAGGTAGGTCAGAGAGCA-3’, and
an expected 200 bp amplification product was generated. SYBR Green-I
RT-qPCR assays were performed using Novozymes Ultra SYBR Mixture
(Vazyme), according to the manufacturer’s instructions.
Two linear standard curves were constructed using tenfold dilutions of
type II and type IV PPRV standard plasmids, using a dilution
concentration of 1.0×108-1.0×102copies/μL. All samples were in triplicate and tested independently. The
equations for the type II and IV PPRV standard curves were y = -3.1734x
+ 31.044 and y = -3.1358x + 32.956, respectively. Standard curves
analysis demonstrated that when the copy numbers were in the range of
108 and 102 copies/µL, the
amplification efficiency of type II and IV PPRV was 106% and 108%,
respectively. The correlation coefficient R2 value for
the linear regression equation of type II and IV PPRV was 0.9981 and
0.9984 (Fig. 1A and 1B). In addition, the detection limits for type II
and IV PPRV were 10 copies/µL. The melting curves for both type II and
IV PPRV were single peaks and melting temperatures (Tm) were 83.39℃ and
81.92℃, respectively (Fig. 1C).
To verify the specificity of the assay, type II PPRV, Orf virus (ORFV),
goat poxvirus (GTPV), and Foot-and-mouth disease virus (FMDV) genomic
cDNA or DNA, and type IV PPRV plasmids were used as templates and
amplified using the conditions described above. Only PPRV (type II and
IV) was amplified in these samples and no specific peaks appeared in the
other samples (Fig. 1D). This indicated that the established real-time
RT-qPCR assay was specific for PPRV and does not cross-react with other
pathogens.
Standard plasmids (type II and IV of PPRV) were diluted
(107,105,103copies/µL) to assess intra- and inter-assay reproducibility. And the
coefficients of variation (CV) of CT values were all less than 5%.
Specifically, for type II PPRV plasmids, the CV of the inter- and intra-
assays was ranged from 0.06% to 0.49% and 0.07% to 6.09%, and for
type IV PPRV plasmids, the CVs were ranged from 0.06% to 0.30% and
1.03% to 6.42%, respectively. These results indicate that the
real-time RT-qPCR assay had good reproducibility.
To evaluate the assay’s potential performance in clinical setting, we
calculate the melting temperature in the qRT-PCR target region of all
the PPRV lineage II and IV strains(9 lineage II strains, 23 lineage IV
strains isolated from foreign countries, and 37 lineage IV strains from
Chin) (Table 1). The average, median and standard deviation melting
temperature of lineage II (IV) amplified area is 82.34℃ (80.97℃), 82.30
℃ (81 ℃) and 0.19℃ (0.14℃), respectively. Furthermore, there was no
significant difference between lineage IV strains isolated from China
and foreign countries. The DNA denaturation temperature diversity
between the two lineages is about 1.3℃ which can be easily reflected
after MCA, it indicates this assay can efficiently detect and
differentiate N75 and PPRV lineage IV isolates when used in the future.
The stability of the DNA duplexes can be affected by the GC content and
length of the PCR products. Thus, the fluorescence signal of the SYBR
Green I was different and could be used to specifically identify PCR
products by MCA. It is very similar to high-resolution melting (HRM) but
more convenient(Hou and You 2018).
At present, there are many detection methods for PPRV, which are mainly
designed to detect all PPRV strains and can not distinguish between
field PPRV strains and vaccine strains (Polci et al. 2015; Mahapatra et
al. 2019; Lucas et al. 2020). The real-time RT-PCR method designed by Li
et al. (Li L. et al. 2016) could specifically detect lineage IV PPRV,
while the other three lineages could not be detected which limits of
detection was found to be 10 copies/reaction. Yang established two rapid
detection methods for PPRV, real-time and lateral flow strip RT-RPA. The
sensitivity of both was as low as 100 copies/reaction at 40℃ and as low
as 150 copies/reaction at 39 ℃, respectively (Yang et al. 2017). All
these methods can detect PPRV rapidly and effectively, but cannot
distinguish between the PPRV vaccine strain and the prevalent strain in
China. In this study, we describe for the first time that only uses one
pair of primers to distinguish lineage II and IV of PPRV based on the
MCA and without reducing sensitivity. The sensitivity of the method is
10 copies/reaction. Furthermore, it does not have cross-react with other
viruses such as ORFV, GTPV, and FMDV.
In conclusion, in this study, we developed an MCA-based SYBR Green I
RT-PCR assay to effectively detect and differentiate lineage II and IV
of PPRV. The detection method is
accurate, rapid, sensitive, reproducible, and easy to perform.
Therefore, this detection method can be used for subsequent
epidemiological investigations and PPR eradication.
Acknowledgements This study was supported by the National
Natural Science Foundation of China (No. 32172832, No. 32000109),
Shanghai Sailing Program (20YF1457700), the China Postdoctoral Science
Foundation (No. 2019M660885, No. 2021T140718), the Central
Public-interest Scientific Institution Basal Research Fund (2021JB08).
Conflict of interest The
authors report no declarations of interest.