**MUMPS virus standard RT-PCR overview**

####### Use of trade names or commercial sources is for identification only and does not imply endorsement by CDC.

[The protocol provided is for use to develop and validate a mumps
detection RT-PCR test as a research tool. CDC cannot guarantee the
performance of any reagents or methods. Results should not be described
as having been obtained using a CDC test and no indication should be
given that CDC has validated the test.]{.underline}

**Primers for Mumps** (PCR product will be 675 bp) :

SH1, AGT AGT GTC GAT GAT CTC AT

SH2R, GCT CAA GCC TTG ATC ATT GA

  ---------------------------------------------------------------------- --
  Primer sequences are from: Jin L, Beard S, Brown DWG. Genetic          
  heterogeneity of mumps virus in the United Kingdom: identification of  
  two new genotypes. J Infect Dis 1999;                                  
  180:829![](media/image1.png){width="8.333333333333333e-2in"            
  height="5.0694444444444445e-2in"}33                                    

  ---------------------------------------------------------------------- --

CDC has prepared aliquots of mumps virus for distribution as a mumps
virus control (0.5 ml) consisting of a cell culture lysate of a wild
type mumps virus grown in Vero cells.

# [Cycling Parameters for Superscript]{.underline}

SUPERSCRIPT RT-PCR PROTOCOL

This is a standard RT-PCR reaction. This is a one-tube reaction, so
there is a minimum of specimen handling. [Some points to
consider]{.underline}:

1\. Controls.

**Positive control** should be RNA extracted from cells infected with
mumps virus. It is best to use cells infected with a well-characterized
strain of mumps, belonging to a genotype not associated with the
outbreak. In addition, the use of vaccine virus as a control is not
recommended since in an outbreak setting some cases may be referred for
testing who have been recently vaccinated. If there is a question of
contamination, this will allow sequence studies to determine the
possible source of the virus.

**Negative controls** should include RNA extracted from uninfected cells
as well as a control with water substituted for RNA. When doing large
numbers of RNA extractions, it is a good idea to include an extraction
control. This mock extraction will show that reagents for RNA extraction
have not been contaminated by template.

2\. The **30-second extension time** shown below is for templates less
than 1000 bp in length. For longer templates, it is advisable to
increase the extension time.

3\. **Avoid template contamination**. Use dedicated equipment, rooms and
hoods for all pre-PCR procedures. Post amplification analysis and
processing should be performed in separate rooms using dedicated
equipment. Do not share equipment (including lab coats) between pre-PCR
and post-PCR procedures. Use filter tips for all pipetting of pre-PCR
procedures and for setting up RT-PCR reactions.

4\. We usually use 5 ul of **RNA** per reaction. Most of the extraction
protocols yield 40-50 ul of RNA. We usually extract RNA from one 25
cm^2^ flask of infected cells or from 100-200 ul of original viral
sample. The volume of RNA can be increased, but this will not improve
the sensitivity.

**MATERIALS:**

1.  Superscript kit (Invitrogen CAT# 10928-042)

2.  RNasin (pancreatic RNase inhibitor)

3.  Ice bucket

4.  Autoclaved PCR tubes (0.2ul, thin-walled)

5.  Forward Primer (20uM working dilution prepared in RNase-free water)

6.  Reverse Primer (20uM working dilution prepared in RNase-free water)

INSTRUCTIONS:

1.  Thaw all kit reagents except enzymes, vortex and place on ice. Keep
    enzymes (Superscript and RNasin) on ice at all times. Allow RNA
    samples to thaw on ice and keep on ice while you are setting up the
    reactions.

2.  Label appropriate number of 0.2 ml thin-walled, reaction tubes and
    place in pre-chilled metal cooling rack. Keep cooling rack on ice
    for entire protocol.

3.  Add appropriate volumes (see worksheet in appendix) of reagents 1
    through 5 to a pre-chilled 1.5 ml Eppendorf tube. Vortex and keep
    tube on ice.

4.  Allow time for pre-mix contents to chill, then add reagents 6 and 7
    to pre-mix tube. Vortex and chill briefly on ice.

5.  Dispense pre-mix (see worksheet) to each reaction tube.

6.  Using a new, clean pipette tip for each transfer, add RNA to each
    tube and close the cap.

7.  Spin the tubes briefly (10,000 rpm for 1 minute) in a chilled
    microcentrifuge and immediately return the tube to the metal cooling
    rack.

8.  While tubes are spinning start the appropriate file on a 9700
    thermocycler. Use the "view" command to make sure that you are
    running the proper conditions. When the block temperature reaches 55
    C, hit the pause button. With the instrument paused, transfer the
    reaction tubes from the metal cooling rack to the heat block of the
    thermocycler. Close the cover and press the resume button.

Appendix

SUPERSCRIPT RT-PCR REACTION WORKSHEET:

Date:­\_\_\_\_\_\_\_\_\_\_\_\_\_\_
Operator:\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

  ----------------------------- ----------- ---------- -------- ------- -------
  Component                     \[Final\]   Vol/rxn    \# rxns  Total   Added
                                Conc.                  plus 1   vol.    Y/N

  1\. 2X Superscript Reaction   1X          25 ul                       
  mix                                                                   

  2\. 5mM MgS04                 0.8mM       8 ul                        

  3\. Forward Primer (20 uM)    0.2 uM      0.5 ul                      

  4\. Reverse Primer (20 uM)    0.2 uM      0.5 ul                      

  5\. ETF water (14.5 -- vol                \_\_\_ul                    
  RNA)                                                                  

  Vortex and place on ice                                               
  before adding enzymes                                                 

  6\. Superscript Enzyme                    1 ul                        

  7\. RNase Inhibitor           20 U.       0.5 ul                      
  (optional)                                                            

  Vortex and dispense pre-mix                                           
  (50ul -- RNA                                                          
  vol)\_\_\_\_\_\_\_ into each                                          
  chilled reaction tube, then                                           
  add RNA samples                                                       

  8\. RNA                                   \_\_\_ ul                   
  ----------------------------- ----------- ---------- -------- ------- -------

Forward Primer:\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Reverse
Primer:\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

## Total reaction volume: 50 ul

Pre-mix dispense volume (50ul -- vol RNA):\_\_\_\_\_\_\_\_