SMRV PCR Diagnostic

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Expert SMRV testing in cell lines

Detection of SMRV in cell lines

Test specifications

Test method

Result report, confidentiality

Sample material

Sample preparation and shipment

Precautions

Detection of SMRV in Cell Lines 

SMRV (squirrel monkey retrovirus) was isolated from a lung tissue of a squirrel monkey (Saimiri sciureus) in 1977. It was characterized as an endogenous type D retro virus. Infection of other organisms is not described but contamination of numerous vertebrate cell systems from human, dog, mink, and other monkey origin demonstrated its wide range in vitro.

Regular inspections of German laboratories report a wide-spread contamination of vertebrate cell lines with SMRV. The German Central Commission for Biological Safety (ZKBS) is afraid that the exchange of cell lines between laboratories may have enhanced the spread of SMRV.

ZKBS recommends testing of all used cell lines for SMRV by PCR. The results have to be reported to German officials and are requested during routine inspections.

Test specifications 

Detection of SMRV after DNA extraction and in case of a positive result RT-PCR after RNA extraction with SMRV specific primer sequences targeting the gag and env genome regions. For in process control the expression of beta-actin is tested as a reference.

Test method 

A) PCR based detection of SMRV specific sequences in the genome of the host cells (SMRV screen, Cat.-No. 41-1005)

The detection of proviral DNA in the genome of the cells is performed in the first step by DNA extraction using a commercial extraction kit (e.g. Qiagen Blood Mini) and in the second step by amplification of specific gag and env sequences in separate PCR tubes. The cell line can be confirmed free of SMRV if both PCR are negative and an amplification of the in SMRV control plasmid is detectable. A positive result will show an amplification of either env or gag or both sequences.

(B) Detection of replicated virus (SMRV direct detection, Cat.-No. 41-4005)

In case of a positive test (A) the cell culture supernatant will be tested by RT-PCR for replicated virus after RNA isolation and RT-PCR. RNA will be isolated from the sample material by using a commercial RNA extraction kit (e.g. QIAamp Viral RNA Mini Kit). DNA will be digested and RNA transcribed into cDNA. The PCR described under (A) will identify any SMRV particles. A positive result will show an amplification of env, gag, or both sequences but not with the untranscribed RNA extract. An amplification of the in process control will be detectable in case of missing SMRV particles in the culture medium.

Result report and confidentiality  

On request the result report can be send via FAX or as a PDF by email as soon as the results are available. A printed hard copy on letter head including a picture of the gel electrophoresis will be send by regular mail.

All customer and result information will be treated strictly confidential. A confidentiality agreement is available on request. All customers have the opportunity to audit the service lab. All result reports are stored on file for a time period of 5 years. Samples are stored for at least 6 months.

Sample material  

Cells including cell culture supernatant.

Sample preparation and shipment  

Please abrade adherent cells and resuspend them in 400 µl of culture supernatant. Transfer the suspension in a sterile, tightly closed vial (e.g. Eppendorf reaction tube). In case of suspension cell lines please centrifuge 1.4 ml of the homogenized culture for 5 min at 500 x g. Resuspend the pellets in 400 µl of culture medium. All cultures shall be tested at 90 to 100 % confluence. Label the vial permanently and easy readable.

For SMRV screen only!!! Please boil the sample material for 10 min at 95 °C.

Send the sample material together with the signed Diagnostic Order Form available here by over night courier according to the local and international shipping instructions (e.g. UN3373, P650) to:

Minerva Biolabs GmbH
- Labor -
Landhausring 7 (ehem. Brebacher Weg 15, Haus 27)
12683 Berlin
Germany

For SMRV Screen only!!! please send the samples at room temperature.
For SMRV detection of replicated SMRV only!!! please send the samples with cool packs or on dry ice.

Precautions  

Minerva Biolabs does not accept sample material of biological safety level 3 and 4. The offered service is not applicable for human testing or any other human use. For quality assurance and research use, only.