Murine Norovirus Mnv Detection From Mice Faecal Sample Biology Essay

Published:

Real time PCR is a technique used to monitor the progress of a PCR reaction in real time and to quantify relatively small amount of PCR product.

Principle: The principle of real-time PCR is a standard PCR reaction carried out in the presence of a dye, which fluorescence when intercalated in the DNA helix. The fluorescence will increase as the amount of the PCR product increases and quantified after each completed PCR cycle. This is the simplest form of real-time PCR called Cyber green in which the flurescent dye can attach to any double stranded DNA. Other sorts of real-time PCR usually contain an extra short oligo which can attach to the amplicon, e.g. Taq Man, Molecular Bacon, Scorpion. Compared to traditional PCR, the master mix of these real-time PCR contains an extra oligo, namely, probe. This probe is a short oligo whose one end is marked with Reporter and other end is marked with Quenchener. The principle of this reaction is based on Fluorescent resonance energy transfer (FRET), meaning that when DNA polymerase is making new strand of DNA, it depolymerases the probe sitting on the DNA template. As a result, Reporter Dye separates away from Quenchener, starts exciting which can be detected by real-time PCR machine.

Lady using a tablet
Lady using a tablet

Professional

Essay Writers

Lady Using Tablet

Get your grade
or your money back

using our Essay Writing Service!

Essay Writing Service

Application: Real-time quantitative PCR allows the sensitive, specific and reproducible quantitation of nucleic acids. Real-time quantitative PCR has revolutionized the field of molecular diagnostics and the technique is using in a rapidly expanding number of applications. [2] Such as:

Quantitative mRNA expression studies

DNA copy number measurements in genomic or viral DNAs.

Allelic discrimination assays or SNP genotyping.

Verification of microarray results

Efficacy of a Drug therapy

DNA damage measurement.

Forensic lab etc

Murine norovirus (MNV): Noroviruses are a group of genetically related and antigenically diverse viruses that can infect both humans and animals.[3]

Norovirus of the family Caliciviridae contains a large number of single-stranded, positive-sense RNA viruses that infect vertebrates, and strains have been identified in humans, cattle, swine, and (most recently) mice.[4]

Most norovirus genomes range from 7.7-7.9 KB and contain three highly conserved open reading frames (ORF). Human Norovirus (HNV) strains are the predominant cause of non-bacterial gastroenteritis worldwide and primarily transmitted through the faecal-oral route, usually by the consumption of contaminated food or water.[6]

Aim of the study

The aim of the study is the detection of Murine Norvirus (MNV) in mice faecal using real time-PCR.

Materials & Methods

Protocol used for the MNV detection in the SVA diagnostic laboratory:

i. General

Pool samples

Dilute samples

Cotton swab to TE Buffer

Homogenize organ tissue

Procedure: There were 62 samples, each sample has five mice faecal; so 300 mice used to collect the samples. Faecal was mixed using the cotton swab and then it was putted in small tube containing 850µl of TE buffer solution. Mixer homogenized for 10-15minutes.

ii. Sample preparation

Procedure: 65µl of TE buffer and 10µl of Prokinase-K and 25µl of the sample added in a small tube and mixed using the vibrator.

iii. RNA/DNA extraction

Magnatrix extraction robot for DNA and RNA separation in overnight

Viral NA- (Nor Diag)

Magnetic Beads

Procedure: The samples were putted in the Magnatrix extraction robot overnight for the separation of the DNA and RNA.

iv. Real time PCR (reverse transcription) for MNV (in house)

Master Mix:

5x RT-PCR buffer ------ 3,00 µl

MgCl2 (25mM)---------- 0,75 µl

dNTP (10mM)---------- 0,60 µl

Probe (10mM) --------- 0,30 µl

Primer both F and R----0,30 µl each

H2O------------------------7,09 µl

RNA-Guard---------------0,06 µl

Enzyme mix -------------- 0,60 µl

Procedure: Master mix was prepared by mixing those and 13 µl of master mix was added in the PCR tubes (1-9 mark). Then 2 µl of the sample added. Two positive controls added in the PCR tube. Then the PCR tube centrifuged to get rid of the air bubble.

v. PCR reaction

Step 1: 500C for 30 min

Step 2: 950C for 15 min

Step 3: 950C for 15sec and 60C for 1min (45 cycle)

Lady using a tablet
Lady using a tablet

Comprehensive

Writing Services

Lady Using Tablet

Plagiarism-free
Always on Time

Marked to Standard

Order Now

Procedure: PCR tube putted in the ABi 7500 fast Machine for the PCR reaction and the machine was set for three steps; step-1 was 500C for 30 min, step-2 was 950C for 15 minutes and step-3 was for 45-cycle run of 950C-15sec with 600C-1min.

vi. Analyze the results:

Set threshold

Ct- values

Results

The figure 1 shows the real time PCR results for the murine norovirus. There were 2 positive controls and 62 mice faecal samples for the test. All the samples (62 faecal samples from laboratory animals, mice) were negative in this real-time PCR.

The threshold was set to 100000 and at that threshold; Ct-value for the positive control was 26. All the negative controls were negative.

Fig 1: Rt-PCR figure after the PCR reaction completed in ABi 7500 fast Machine.

Discussion

Currently, there is no cell culture or small animal model available for studying human norovirus strains, thus presenting a need to find an appropriate model.[6]

Murine norovirus (MNV) is a viral agent newly identified in laboratory mice and a large number of genetically diverse MNV strains have reported to date. A broadly reactive TaqMan-based real-time reverse transcription (RT)-polymerase chain reaction (PCR) assay was developed for MNVs.[7]

The prevalence of Murine norovirus (MNV) in this set of samples was zero, although MNV has been found at high prevalence in other laboratory mouse colonies.

Murine Norovirus will potentially become a useful model for the human noroviruses, the human virus may foretell what is to come for MNV. The genus Norovirus is in the family Caliciviridae which also includes the genera Vesivirus, Sapovirus, and Lagovirus. Some individuals can develop chronic disease however most cases are acute disease of 1-2 days duration. [8]

Murine norovirus (MNV) has recently been recognized as a widely prevalent viral pathogen in mouse colonies and causes disease and mortality in mice with impaired innate immunity. The effects of MNV on models may occur early after infection and may be short-lived, but the time of introduction and exposure of a particular mouse to MNV may not be known during natural transmission. Endemic infections may have a different pathogenesis than acute infections, so it is crucial that future studies include investigations into control and prevention along with identification of the effects of chronic, persistent infections.[4]

In addition, many advances in understanding mechanisms of calicivirus replication have come from studies using related animal caliciviruses, such as FCV. The recently developed replication system for human noroviruses may be used to directly compare norovirus and calicivirus replication at the cellular level.[9]