Application of reverse transcriptase nested, hemi-nested And multiplex polymerase chain reaction techniques For the detection of pv 11, pv 3462 and cvs Strains of rabies virus

Sekar, Thangaraj; Mohan, Ganesan Chandra; Karthick, Bala Subramanian; Premkumar, Ananda Arone; Sundaran, Bheeman; Sekar, Balaraman

doi:10.22376/ijpbs.2018.9.3.b280-286

Cited by 1 publication

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References 18 publications

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“…The samples were analyzed for the presence / absence of rabies virus specific genome amplification with the multiplex PCR. 40μl master mix per reaction was prepared with two pairs of primers (NP & MP gene specific primer) and amplified in eppendrof thermal cycler (nexus GSX1) with the following amplification cycle; initial denaturation at 94°C for 5min followed by 94°C for 30s, 59°C for 45s, 72°C for 45s for 35 cycles with final extension at 72°C for 7min [13].…”

Section: Inactivation Of Virus Ensured By Multiplex Pcr (Polymerase Chain Reaction)mentioning

confidence: 99%

Rabies vaccine purification using HPLC, Estimation of residual vero cellular DNA by RT-PCR and potency analysis

Sekar¹,

Premkumar²,

Mohan³

et al. 2019

Madridge J Vaccines

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The rabies is a highly fatal, viral zoonotic disease can be preventable by effective vaccination. The anti-rabies vaccine prepared by cell culture technology are safer than the nerves tissue vaccine. The PV-11 strain of rabies virus was propagated in vero cells and subjected for purification using HPLC and potency analysis in this study. The viral harvest were concentrated up to 10X by tangential flow ultra-filtration with 100 kDa MWCO cassettes. The vero cell DNA in the concentrated material were removed by protamine sulfate precipitation and the virus inactivated by β propiolactone. The inactivation of virus was assured through Avirulece test, virus amplification test and PCR. The vero cell DNA in different stages of vaccine preparation were quantified by real time -PCR. It was estimated that maximum of 94.85 pg in viral harvest, 119.4 pg in concentrated material and 23.6 pg in final purified vaccine. The rabies viral protein was detected through HPLC analytical column in comparison with the international standard reference vaccine and purified by preparative column. Further dot blot hybridization assay was performed to quantify the rabies viral protein. The purified preparation was sterile filtrated through 0.22µ filtration, formulated with two different preservative and stabilizers. The final vaccine was lyophilized in vials under aseptic condition, subjected for NIH Potency analysis in mice model to estimate the potency unit of our vaccine preparation and the potency units were calculated using the probit analysis software in comparison with international standard reference vaccine. The potency unit of 4 different samples from two different formulations were measured. The potency unit of batch 1 formulations were 5.72 and 6.34 IU/ single dose and batch 2 formulations were 2.80 and 2.89 IU/ single dose. Both the vaccines passes the minimum requirement of 2.5 IU/single dose.

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Section: Inactivation Of Virus Ensured By Multiplex Pcr (Polymerase Chain Reaction)mentioning

confidence: 99%

Rabies vaccine purification using HPLC, Estimation of residual vero cellular DNA by RT-PCR and potency analysis

Sekar¹,

Premkumar²,

Mohan³

et al. 2019

Madridge J Vaccines

Self Cite

View full text Add to dashboard Cite

show abstract

Application of reverse transcriptase nested, hemi-nested And multiplex polymerase chain reaction techniques For the detection of pv 11, pv 3462 and cvs Strains of rabies virus

Cited by 1 publication

References 18 publications

Rabies vaccine purification using HPLC, Estimation of residual vero cellular DNA by RT-PCR and potency analysis

Rabies vaccine purification using HPLC, Estimation of residual vero cellular DNA by RT-PCR and potency analysis

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