Comparison of Real-Time reverse transcription PCR (RT-qPCR), Enzyme-linked immunosorbent assay (ELISA), and Immunohistochemistry (IHC) for detection of persistent infection with Bovine Viral Diarrhea Virus in cattle.
This is a video presentation discussing my research project, Comparison of Real-Time reverse transcription PCR (RT-qPCR), Enzyme-linked immunosorbent assay (ELISA), and Immunohistochemistry (IHC) for detection of persistent infection with Bovine Viral Diarrhea Virus in cattle.
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[00:00:01.410]Hello, my name is Allie Johnson. Today.
[00:00:03.780]I will be discussing my
project the comparison of time,
[00:00:06.630]reverse transcription PCR, and I'm linked.
[00:00:09.360]Immunosorbent a se and
detection of persistent
[00:00:13.500]infection with bovine viral
diarrhea virus in cattle, bovine,
[00:00:18.870]virus causes bovine viral diarrhea in the
form of acute or persistent infections
[00:00:22.860]in cattle persistently infected cattle
may show little to no clinical signs,
[00:00:27.090]but our liability to the herd as they
may continually shed the virus and cause
[00:00:30.930]acute infections and other cattle,
[00:00:33.000]acute BVD presents with
numerous clinical science,
[00:00:35.640]including the potential for abortion
because of the economic consequences of
[00:00:40.020]maintaining persistently infected
animals control programs,
[00:00:43.080]focus on identification and elimination
of these animals book screening of
[00:00:47.820]seizing pool that you're not just has
been developed to determine herd status,
[00:00:51.330]which should be followed by further
testing to differentiate those individuals
[00:00:55.140]with acute or persistent infections,
[00:00:58.140]diagnostic testing utilized for this
purpose includes real time reverse
[00:01:02.850]polymerase chain reaction enzyme
linked immunosorbent se and anti BBD
[00:01:09.420]The goal of my project was to compare
these testing methods and determined which
[00:01:12.810]of these provided would detect BVDV
with the most reliability figure.
[00:01:17.790]One that you see on the right displays,
[00:01:20.190]the effects of having a PI calf
shedding the virus within herd.
[00:01:23.640]The virus may be transmitted through
any bodily secretion to the other herd
[00:01:26.850]members with the implications,
[00:01:28.560]the lead to economic loss
and decreased productivity.
[00:01:32.610]In figure two, you can
see a series of images.
[00:01:35.880]The Tufts of images we see have diffused
ulceration of the skin and mucosa
[00:01:40.530]caused by BVB images,
[00:01:42.360]three and four in the center
show ulceration of the
esophagus and to Juno image
[00:01:46.890]five on the bottom left shows myocardial
degeneration and necrosis while image
[00:01:51.180]six on the bottom, right?
[00:01:52.200]Is this a cross section of tissue
from image five showing positive BVDV
[00:01:57.150]staining from IHC, which is
denoted by the red coloring.
[00:02:02.130]Your notch tissue biopsy samples
were collected from 5,580 cattle and
[00:02:07.050]sent to the Nebraska veterinary
diagnostic center. In January of 2020.
[00:02:11.070]These samples were processed by dispensing
two milliliters of phosphate buffered
[00:02:15.360]saline into each tube
containing a small ear notch.
[00:02:18.750]The PVS from each individual you're
not samples were pooled into 117
[00:02:23.670]pools. Each containing samples from
40 individuals to be tested for PCR
[00:02:29.160]extraction was performed on the pools.
[00:02:30.900]Following the lowest count
method of Magmax extraction
using the Magmax pathogen
[00:02:35.640]RNA DNA kit, since BVDV contains its
genetic material as single-stranded RNA,
[00:02:41.010]the real-time reverse transcriptions
includes an initial step of converting the
[00:02:44.610]viruses Arning into a DNA template.
[00:02:46.950]The real-time reverse transcription PCR
was done utilizing the Qiagen one-step
[00:02:53.340]The primers and probes employed are
listed Bio-Rad artsy thermocycler was used
[00:02:58.050]with three steps listed, which
completed a total of 40 cycles.
[00:03:01.990]Master mix was prepared according to the
protocol for Qiagen one-step RTPCR kit
[00:03:07.480]and sign linked immunosorbent to say, or
Eliza was also performed on your notch.
[00:03:11.290]So buffer for each
sample using the BVDV P I
[00:03:16.000]X two antigen Tesco and it's manual
detection of the antibodies or antigens
[00:03:20.710]occurs through a color reaction that
is measured by optical density of the
[00:03:25.600]Immunohistochemistry or IHC was used
to confirm the positive samples from
[00:03:30.340]Eliza and PCR testing.
[00:03:32.170]I see analysis of the samples involved
detecting antigen is present in formal
[00:03:36.260]Olympics. You're not just new skin.
[00:03:37.840]Biopsies slides were prepared
following standard IC procedure,
[00:03:42.640]utilizing an M antibody against BVDV.
[00:03:45.760]As the primary antibody slides were placed
in the benchmark ultra instrument and
[00:03:49.780]subjected to the UltraVIEW red
protocol detection of the virus and
[00:03:54.400]IFC is observed through the reaction that
occurs between antigen and antibodies
[00:03:58.840]as cross-linking takes place.
[00:04:00.340]Same collects in a red coloring is
shown into gaining a positive sample.
[00:04:04.930]So she's sections were then examined by
a trained pathologist to determine PI
[00:04:13.030]The 117 pools were tested using PCR
to determine her status and of the
[00:04:18.010]117 pools. There were four positive pools,
[00:04:21.160]including pools nine 30, two 39 and 79.
[00:04:26.110]And they're respected. CT values
are shown in figure three.
[00:04:30.010]Eliza testing was used to
verify results from PCR.
[00:04:33.490]The Eliza results were
interpreted using SP values,
[00:04:36.280]which were calculated based on the
obstacle density values of the samples.
[00:04:39.970]Figure four depicts the Eliza results,
[00:04:42.010]showing the P values for those individuals
that tested positive from each of the
[00:04:46.990]There were three individuals samples
positive and pool nine in one individual
[00:04:50.710]positive sample and pools,
[00:04:52.450]30 to 39 and 79 as fee values
of greater than or equal
[00:04:57.280]to 0.15 were considered positive
IHC was used to validate these
[00:05:03.250]It exposed to false positives detected
by Eliza to have the positive samples
[00:05:07.510]from pool nine,
[00:05:08.890]sample nine dash two and nine dash
three were negative for BVDV and
[00:05:14.410]The other samples from
pools 32 39 and 79 were as
[00:05:19.270]well as nine dash one from pool
nine were confirmed to be positive.
[00:05:24.640]Overall IHC is considered
to be the gold standard.
[00:05:27.280]So it was used as baseline to distinguish
true positive samples from false
[00:05:31.420]positives. Eliza correctly identified
for the positive BVDV animals,
[00:05:36.340]but did have two false
positives with this.
[00:05:38.680]The sensitivity of Eliza was
calculated to be 100%. However,
[00:05:43.870]the specificity was 98.9% PCR
and IC both performed with a
[00:05:48.850]sensitivity and specificity of 100%.
[00:05:52.600]The Kappa coefficient was calculated
using jump statistical software between
[00:05:57.290]Eliza and IFC, which
came out to be 0.7949,
[00:06:02.000]which displays substantial
agreement indicating that
Eliza may also be useful to
[00:06:05.900]distinguish disease status
[00:06:09.170]In conclusion with the potential to cause
high levels of financial loss due to
[00:06:13.130]its severity of clinical signs
and effects on productivity.
[00:06:16.280]It is imperative to work towards control
and eradication of BBD real time,
[00:06:21.290]reverse transcription PCR, IHC.
[00:06:24.410]And I have demonstrated the ability to
detect the virus with elevated accuracy
[00:06:29.090]after reviewing results
from 5,580 year not samples,
[00:06:33.320]it can be determined that a
combination of PCR and IC may provide
[00:06:38.240]the highest levels of
specificity and sensitivity.
[00:06:41.870]Both resulting in 100%.
[00:06:44.120]PCR may be used to determine herd
status followed by IHC to determine
[00:06:48.890]persistently infected animals, which may
be contributing to ongoing infections.
[00:06:54.920]My references utilized
can be seen on the right.
[00:06:57.530]I would like to thank the Nebraska
veterinary diagnostic center and its staff
[00:07:00.980]for processing and testing these samples
and for allowing the results to be
[00:07:04.520]compared. I would also like
to thank Dr. Dewan loin, Dr.
[00:07:08.120]Jay Dustin Lloyd and Dr. Bruce Broderson
for all their help and guidance.
[00:07:12.890]Thank you for taking the time
to listen to my presentation.
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