Evaluation of a real time pcr to simultaneously detect and differentiate virulent and non-virulent Newcastle disease virus



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Objective 3:

To technology transfer the newly acquired NDV PCR test to Biosecurity Sciences Laboratory (BSL, Queensland Department of Agriculture, Fisheries and Forestry, DAFF)

All information regarding the test of choice and the conditions for use were provided to BSL. A small change in the running conditions were needed compared with the DPI Vic laboratory given that the QLD laboratory use a different Real time PCR platform (Corbett compared to Applied Systems at DPI Vic). Therefore the appropriate reagents were used to optimise the test at the QLD laboratory.

There are long delays in receiving the probe after ordering due to the locked nucleic acid requirement which complicates the manufacture. Therefore, DPI Vic sent primers and probes to BSL whilst their stocks are on order. This had the added advantage of standardising an additional component of the testing given that identical batches of primers and probes were used at both laboratories.

BSL tested a total of 26 samples in the newly transferred virulent/avirulent NDV PCR test (refer to Table 5). This included a range of virulent (n=8), avirulent (n=15) and negative (n=3) NDV samples. As expected, none of the three Class I NDV or three negative samples were detected in the virulent/avirulent NDV PCR. Of the 20 Class II APMV-1 samples tested, 18 samples were correctly detected in the virulent/avirulent multiplex qPCR, and two samples were not detected in the virulent/avirulent multiplex qPCR. These results very much reflect the results obtained by the DPI Victoria group, where the virulent/avirulent multiplex qPCR was able to correctly identify the majority of the reference strains, however those with high Ct values (low viral load) were missed. This reflects the lack of sensitivity of the multiplex qRT-PCR on the Corbett instrument. This is in agreement with the observations made on the Applied Biosystem instrument. The lack of sensitivity was also illustrated by the inability of both PCRs to detect the NQC-1, a highly dilute RNA from the Deans Park isolate, a virulent NDV.



Objective 4:

Undertake phylogenetic analysis of pathotyping sequencing results to show relationships of clades
It was planned to undertake phylogenetic analysis for any virulent strains detected during the course of this study. However, despite testing only avirulent viruses were detected and therefore the phylogenetic comparisons could not be completed.

Table 5: BSL (Queensland DAFF) Fuller Real time PCR (ARP/VRP) results using a standard panel of APMV-1 positive samples. (Note that the same batch of primers and probe were used for both DAFF and DPI Vic, however a Corbett Rotor-Gene PCR machine was used at BSL)




Samples

Class

Vir/Avir

Virulent/Avirulent APMV-1 qPCR

ARP

VRP

Nepal Kaski

II

V




31.07

Nepal Kailali

II

A

32.48




Nepal 1111

II

V




23.16

Nepal 1114

II

V

ND

ND

VN 1116

II

V




21.21

F Strain (vaccine)

II

A

29.95




WA 1886

I

A

ND

ND

WA 4317

II

A

24.65




WA 5332

II

A

22.34




WA 4359

II

A

24.90




WA 4386

II

A

22.31




WA 4405

II

A

27.21




5760

II

A

43.17/-




APMV1 Shep 1109-14-4303

II

V?




31.17

Ibis/Qld 0702-14-1242

I

A

ND

ND

Ck/Nepal/Kaski 0804-18-0210

II

V




39.14

F strain

II

A

29.64




Negative

-

ND

ND




Negative

-

ND

ND




V4

II

A

28.89




Deans Park

II

V




38.66

V4

II

A

28.09




A/Ch/NSW/Aust/CV10-1004-3/2010/H10N7

-

ND

ND




NDV NQC-1

II

V

ND

ND

116603

I

A

ND

ND

NDV V4

II

A

31.87



ND Not detected



Conclusions

This project has seen the successful introduction of a new qPCR test at DPI Victoria capable of differentiating virulent from avirulent APMV-1. A test such as this is invaluable for use with diagnostic avian samples because the current variety of tests available for screening for APMV-1 can only provide a positive or negative result for the presence of APMV-1 regardless of whether or not the strain of APMV-1 is virulent or avirulent. This is further complicated by the extensive use of the NDV vaccine in which case samples from vaccinated birds appear positive in the screening tests due to the avirulent V4-like strain of NDV used in the vaccine. A test capable of confirming that those screening test positives are from avirulent APMV-1 would therefore be a huge advantage.

Whilst duplicate samples were not tested at both laboratories, the transfer of technology to the Biosecurity Sciences laboratory in Queensland (DAFF) was successful. Both the Corbett and Applied Biosystems instruments appear to have performed similarly, however identical samples would need to be compared to confirm any differences in the detection limits between the platforms employed. Furthermore, on both machines two virulent APMV-1 were not detected, underlining the lower sensitivity of the multiplex avirulent/virulent qRT-PCR compared to the Kim/Wise multiplex qRT-PCR.

The virulent/avirulent multiplex qPCR developed by Fuller et al. (2009), was chosen as the best alternative of a test capable of differentiating APMV-1 strains. In our hands, the virulent/avirulent multiplex qPCR worked well with laboratory strains of APMV-1, however it is not as sensitive as other tests available for the detection of APMV-1. Direct comparisons of positive samples tested in the Kim/Wise qPCR and the virulent/avirulent multiplex qPCR showed Ct values approximately 1.5-3 cycles lower (detected earlier) in the Kim/Wise test. In addition the virulent/avirulent multiplex qPCR totally missed some of the Kim/Wise qPCR positives which would lead to the wrong assumption of a false negative result if this test was being used as a screening test. The reason for this is likely to be the complexity of the two probe system and the locked nucleic acid requirement all of which are attempting to hybridise pathogen nucleic acid where there are inherent variations within the area of the genome that the test is designed to.

The virulent/avirulent multiplex qPCR proved to be robust enough to use on infected chicken and pigeon samples with relatively high viral loads. However it did not perform well with wild bird samples that presumably have a much lower viral load and so consequently had a high Ct value in the Wise qPCR.

Therefore in conclusion, we recommend that the Kim/Wise multiplex qPCR or other such qPCR continue to be used as the screening test to detect APMV-1. The new virulent/avirulent multiplex qPCR should only be used on known PCR positive samples in an attempt to determine whether the Class II APMV-1 virus detected was virulent or avirulent. In the case of a weak positive PCR screening result, there is a high likelihood that the virulent/avirulent multiplex qPCR will not provide a result. Preliminary results at this laboratory have shown that introducing a nested PCR format using the F gene PCR of Collins et al., (1993) as a first round PCR prior to conducting the Fuller et al., (2009) virulent/avirulent multiplex qPCR, may resolve some of the issues with the diagnostic sensitivity on samples with a lower level of nucleic acid.



Acknowledgments

We would like to thank Dr Frank Wong and Paul Selleck from the CSIRO Australian Animal Health Laboratories for the provision of virulent NDV samples for use in the development of this test.



References

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Wise MG, Suarez DL, Seal BS, Pedersen JC, Senne DA, King DJ, Kapczynski, Spackman E. (2004). Development of a real-time reverse-transcriptase PCR for detection of Newcastle disease virus RNA in clinical samples. J Clin Microbiology 42:329-338.







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