**Disclaimer**: Use of trade names and commercial sources in this
protocol does not imply endorsement by the Centers for Disease Control
and Prevention (CDC).

**Real-time (TaqMan®) RT-PCR Assay for the Detection of**

**Mumps Virus small hydrophobic (SH) RNA**

***General Comments***

**THESE PROTOCOLS SHOULD NOT BE DISTRIBUTED AND RESULTS OBTAINED WITH
THESE ASSAYS SHOULD NOT BE PUBLISHED OR PRESENTED IN A PUBLIC FORUM
WITHOUT THE EXPLICIT CONSENT OF CDC AND THE UNIVERSITY OF IOWA HYGIENIC
LABORATORY.**

The Mumps virus causes an acute viral illness with symptoms including
fever, headache, muscle aches, tiredness, and loss of appetite, followed
by swelling of salivary glands. The parotid salivary glands (which are
located within the cheek, near the jaw line, below the ears) are most
frequently affected.

***PARAMYXOVIRIDAE* TAXONOMY**

[Subfamily *Paramyxovirinae*]{.underline}

> Genus *Respirovirus*
>
> Sendai virus
>
> Human parainfluenza viruses, types 1 and 3
>
> Bovine parainfluenza virus type 3

Genus *Rubulavirus*

Mumps virus

Human parainfluenza viruses, types 2, 4a and 4b

Simian virus 5

Genus *Morbillivirus*

Measles virus

Canine distemper virus

Rinderpest virus

Genus *Henipavirus*

Hendra virus^a^

Nipah virus

Genus *Avulavirus*

Newcastle disease virus

Avian paramyxoviruses 1-9

[Subfamily Pneumovirinae]{.underline}

Genus *Pneumovirus*

Human respiratory syncytial virus

**Primer/Probe Target:**

[SH Gene (designed at CDC with Primer Express, ABI):]{.underline}

SH61F, GTG ACC CTG CCG TTG CA

SH147R, GTT ATG ATC AGA GAG AGA AGA ATT AGC AAT AG

SH79P2, [FAM]{.underline}-TAT GCC GGC GAT CCA ACC TCC CTT
ATA-[BHQ]{.underline} (probe)

[gi\|9695415\|ref\|NC_002200.1\|](http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=9695415&dopt=GenBank)
Mumps virus, complete genome Length=15384

Primers amplify an 86bp fragment, starting at base number 6249 to 6335
of the mumps viral genome.

The mumps virus genome is a single-strand RNA that contains seven genes
encoding the nucleocapsid (N), phosphoprotein (P), membrane (M), fusion
(F), small hydrophobic (SH), hemagglutinin-neurominidase (HN), and large
(L) proteins. The SH gene encodes a protein of 57 amino acids with
unknown function. The SH gene is the most variable region of the mumps
genome and is used to genotype mumps strains.

[RNAse P RNP (designed at CDC)]{.underline}

Forward AGA TTT GGA CCT GCG AGC G

Reverse GAG CGG CTG TCT CCA CAA GT

Probe JOE-TTC TGA CCT GAA GGC TCT GCG CG-TAMRA

\>![](media/image1.wmf)[gi\|13937783\|gb\|BC006991.1\|](http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=13937783&dopt=GenBank)
![](media/image2.png){width="0.1673611111111111in"
height="0.1673611111111111in"}![](media/image3.png){width="0.1673611111111111in"
height="0.1673611111111111in"}![](media/image4.png){width="0.1673611111111111in"
height="0.1673611111111111in"} Homo sapiens ribonuclease P/MRP 30kDa
subunit, mRNA (cDNA clone. Primers amplify a 64 base pair fragment, base
pairs 309 through 373.

The human RNase P gene (RNP) primer and probe set serves as an internal
positive control to monitor for inhibition of the PCR reaction. This
control cannot by used as a control for reverse transcription (RT) since
it will detect both RNA and DNA.

> **NOTE:** Studies in our laboratory showed that a JOE-TAMRA RNP probe,
> as opposed to JOE-BHQ RNP, allowed for more sensitive detection of the
> mumps virus target.

**Test Specificity:** Mumps virus primer/probe set included in this
protocol has been shown to detect mumps strains D, G, and J-L vaccine
strain. It detected 25 of 25 buccal/oral swabs from culture positive
patients. No positive results were obtained from 50 mumps IgM negative,
culture negative primary specimens.

There was no cross-reaction with viral cultures from human specimens for
parainfluenza virus type 1 (three isolates), parainfluenza virus type 3
(three isolates), respiratory syncytial virus (2 isolates), measles
virus, influenza A, influenza B, adenovirus, HSV, or enterovirus.

**Protocol Use Limitations:**

These protocols were optimized using ABI Taqman® one-step probe RT-PCR
chemistry on a 96-well format thermocycler and have been shown to
produce comparable results on the ABI 7000, 7500, and 7700. **NOTE**: Ct
values obtained using the ABI 7000 are typically higher Optimization of
assay conditions such as primer/probe concentrations may be necessary if
used on other systems such as the Roche Lightcycler^TM^, ABI 7500 FAST,
or Cepheid SmartCycler^TM^ .

The sequence of the SH gene varies between strain of wild-type viruses.
This variability may result in mismatches in the primer of probe
sequences with the target sequence. Therefore, this assay may not be
able to detect RNA from some wild-type strains.

Positive RT-PCR results should be interpreted with signs, symptoms, and
recent MMR vaccine history due to the potential, but not proven,
detection of the vaccine strain in clinical specimens.

**Acceptable specimens:** Preferred specimen is an oral/buccal swab;
oropharyngeal swabs are acceptable. CSF is an acceptable specimen in the
case of suspect mumps meningitis/encephalitis. The nucleic acid
extraction procedure has been validated for CSF. Urine is an acceptable
specimen but has not been validated with this procedure.

[Oral/buccal swab for virus culture:]{.underline} Collect fluid by
swabbing the buccal cavity (the space between the cheek and teeth). The
parotid duct drains in this space near the upper molars.

Massage the parotid gland area just in front of the ear and near the
angle of the jaw for 30 seconds prior to collecting secretions on the
swab. Swab the area between the cheek and gum by sweeping the swab near
the upper molar to the lower molar area. Place the swab in viral
transport medium and leave the swab in the medium.

-   Swab specimens should be collected only with a Dacron® tip and an
    aluminum or plastic shaft.

-   Swabs must be collected in viral transport medium and stored and
    shipped cold (i.e. 4^o^C)

-   Swabs with calcium alginate or cotton tips and wooden shafts are
    unacceptable.

**Rejection criteria:**

-   Specimen not refrigerated

-   Use of improper swab or swab not in viral transport medium

-   Incomplete specimen labeling/documentation

-   Insufficient specimen volume

**Assumptions:** This procedure assumes a basic familiarity with
real-time RT-PCR assays.

**Disclaimer:** Names of vendors or manufacturers are provided as
examples of suitable product sources. Inclusion does not imply
endorsement by the Centers for Disease Control and Prevention (CDC) or
the University of Iowa Hygienic Laboratory (UHL).

**Precautions:**

-   All reactions should be carried out on ice and precautions taken
    against RNase contamination. Work surface and racks should be
    cleaned with 10% bleach solution and equipment such as pipettes and
    centrifuges should be cleaned and decontaminated with products such
    as "Eliminase" or "RNase away" to minimize risk of contamination.

-   Specimens should be processed according to Biological Safety Level 2
    guidelines. Specimens that have the potential to contain live virus
    should be processed in a Biological Safety Cabinet.

-   General Practice

    -   All PCR steps will occur in designated areas for a) reagent
        preparation (No DNA), b) specimen processing, c) DNA
        amplification and d) amplicon detection. Work is directional.

    -   Use aerosol barrier tips, disposable gowns that are dedicated to
        each room, separate micropipettors and equipment for each area.

    -   Reagents: will be purchased with previously documented QC when
        possible. Any solutions made in house will be validated as
        functional and free of contamination prior to use.

    -   Change gloves often.

**Materials**

-   20μl and 200μl adjustable pipettes and aerosol barrier tips

-   0.2ml PCR optical reaction tube strips or 96-well reaction plate

-   optical strip caps or adhesive plate cover

-   sterile, RNase-free, microcentrifuge tubes

-   powder-free gloves

-   microcentrifuge

-   vortex mixer

-   PCR set-up hood

-   real-time PCR detection system

**Reagents:**

-   TaqMan One-Step RT-PCR Master Mix #4309169 Applied Biosystems

-   <http://docs.appliedbiosystems.com/pebiodocs/04310299.pdf>

    -   NOTE: Similar results were

    -   sterile distilled molecular grade water (RNase and DNase free)

    -   SH forward and reverse primers, HPLC purified

    -   SH dual-labeled TaqMan® probe labeled at the 5\'-end with the
        reporter molecule 6-carboxyfluorescein (FAM) and with the
        quencher, Blackhole Quencher 1 (BHQ1) (Biosearch Technologies,
        Inc., Novato, CA) at the 3\'-end.

    -   RNP forward and reverse primers, HPLC purified (40μM) for use as
        internal control

    -   RNP dual-labeled TaqMan® probe labeled at the 5\'-end with the
        reporter molecule JOE and with the quencher TAMRA at the
        3\'-end. The probe is phosphorylated at the 3′ end to prevent
        elongation during PCR. For use as internal control.

    -   Store primer and probe stocks at --20^o^C.

    -   Keep probe stocks protected from light.

```{=html}
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```
-   Nucleic acid extraction: QIAamp® Viral RNA Mini Kit (Qiagen,
    catalogue \# 52906)

<http://www1.qiagen.com/literature/handbooks/PDF/RNAStabilizationAndPurification/FromClinicalSamples/QA_Viral_RNA_Mini/1030454_HB_QA_Viral_RNA_122005.pdf>

Follow manufacturer's instructions for sample extraction.

**NOTE:** Preliminary tests using the MagNA Pure LC Total Nucleic Acid
Isolation Kit (Roche Applied Science, catalogue #03038505001)
demonstrated recovery of detectable mumps virus with a slight increase
in the cycle threshold (Ct). This automated system may be acceptable for
nucleic acid isolation with a slight loss in sensitivity.

**Reagent preparation:**

1.  Keep all reagents on ice during assay set up.

2.  Vortex all primers and probes.

3.  Spin all primers and probes and then place on ice or cold block.

4.  Mix enzymes by pipette or flicking the tube. Do not vortex. Quick
    spin and keep on ice or cold block.

**Controls for RT-PCR**

-   No template controls (NTC)

-   Mumps *in vitro* transcribed SH gene control (strain D, CDC)

-   Mumps infected cell RNA standard (strain D, CDC)

> Make serial ten-fold dilutions of RNA.
>
> Add 2.5 µl from each 10^-6^, 10^-5^, 10^-4^, 10^-3^, and 10^-2^
> dilution into two wells each.

-   Include one negative extraction control for every five specimens.

-   The human RNase P gene (RNP) primer and probe as endogenous positive
    control

**Procedures**

1\. Determine the number of reactions (n) to set up per assay and
prepare excess to allow for controls\
and pipetting errors (n + 1 or 2).

  ---------------------------------- ---------- ----------- --------------
  **PCR Set-up**                      each      **X n+1**   

  samples:                           1                      

  4.175 uL nuclease-free DEPC water  4.175                  

  12.5uL 2X TaqMan One-Step RT-PCR   12.5                   
  Buffer                                                    

  0.625 uL 40X MRI                   0.625                  

  0.5 uL SH61F (15uM, forward        0.5                    300nM final
  primer)                                                   

  0.5 uL SH147R (15uM, reverse       0.5                    300nM final
  primer)                                                   

  0.2 uL SH79P2 (12.5uM, FAM-BHQ     0.2                    100nM final
  probe)                                                    

  0.625 uL RNP-F (1uM, forward       0.625                  25nM final
  primer)                                                   

  0.625 uL RNP-R (1uM, reverse       0.625                  25nM final
  primer)                                                   

  0.25 uL RNP-PR (10uM, JOE-TAMRA    0.25                   100nM final
  probe)                                                    

  **TOTAL 20.0 uL**                  **20**                 
  ---------------------------------- ---------- ----------- --------------

2\. Mix reaction mixtures by pipetting up and down. Do not vortex.

3\. Add 20μl of each master mix into each well of a 96-well optical
plate.

4\. Pipette 5 μl of nuclease free water into the NTC wells, and use 5 μl
of either control or unknown to\
each master mix.

5\. Centrifuge the plate at 500 × g for 30 seconds at 4°C.

**RT-PCR amplification conditions**

1.  Turn on the real-time PCR instrument and allow the system to warm up
    for 30 min prior to use.

2.  Program the thermocycler as follows:

  -----------------------------------------------------------------------
  A\) 48^o^C, 45min

  95^o^C, 10 min

  40 cycles: 95^o^C,15 seconds

  60^o^C, 60 seconds

  B\) Set sample volume to 25ul

  C\) Run FAM-BHQ1 dye layer

  D\) Run JOE-TAMRA dye layer

  E\) Passive reference- ROX
  -----------------------------------------------------------------------

3.  Fluorescence data should be collected during the 60°C
    anneal/elongation step.

**Interpretation/examination**

1.  Set threshold for mumps FAM-BHQ amplification plot and determine
    Cts.

2.  Set threshold for RNP JOE-TAMRA amplification plot and determine
    Cts.

> All clinical samples should exhibit RNP reaction curves that cross the
> threshold line before 40 Ct, indicating the presence of the human RNP
> nucleic acid, indicating that nucleic acid extraction and PCR
> amplification did occur.
>
> Failure to detect RNase P in any of the clinical samples may indicate:

-   Improper extraction of nucleic acid from clinical materials
    resulting in loss of RNA or carry-over of RT-PCR inhibitors from
    clinical specimens

-   Absence of sufficient human cells in sample to enable detection

-   Improper assay set up and execution

-   Reagent or equipment malfunction

3.  A sample is considered positive when a curve crosses the threshold
    before 40 Ct. All laboratories should determine their own equivocal
    range (UHL equivocal Ct ≥ 37).

    a.  All QC must be performed correctly in order to determine that a
        specimen is positive.

    b.  The multicomponent curves must also show true signal in addition
        to the amplification curve.

4.  Mumps virus is not detected in a sample if the RNP Ct is \<40 and
    the SH Ct =40 or undetected.

**ALTERNATE PROCEDURE FOR THE 7900HT**

**Materials Needed:**

-   TaqMan® PCR Core Reagent Kit; 400, 25-µl reactions/kit (Applied
    Biosystems N-808-0228)

-   MultiScribe Reverse Transcriptase; 500 units (Applied Biosystems
    #4311235)

-   RNase Inhibitor x 2; 2000 units@ (Applied Biosystems N-808-0119)

-   Nuclease-free water (Ambion #9937)

-   Quantitative PCR Human reference total RNA (Statagene #750500)

-   MicroAmp optical 96-well reaction plate with barcode (Applied
    Biosystems #4306737)

-   Optical adhesive cover (Applied Biosystems #4311971)

Note: We (CDC) have not used this protocol for multiplex reactions. We
usually run separate viral target and RNase P reactions. However, the
RNase P primers described above will work with these reaction
conditions.

**Protocol:**

1.  Prepare master mix and sample plate worksheets for the number of RNA
    samples to be tested.

2.  Thaw 10X TaqMan buffer, 25 mM magnesium chloride, and dNTPs. Once
    thawed, briefly vortex and centrifuge. Keep components on ice until
    ready to dispense.

3.  Add nuclease-free water, buffer, magnesium chloride, and dNTPs to
    appropriately labeled 1.5 ml microcentrifuge tubes.

4.  Thaw, briefly vortex and centrifuge, before adding the appropriate
    primers and probes. Primer and probe concentrations have been
    optimized.

5.  Briefly centrifuge enzymes (AmpliTaq Gold, MultiScribe RT, RNase
    inhibitor) and keep on ice until ready to dispense

6.  After adding enzymes, invert and briefly centrifuge the master
    mixes. Dispense 22.5 µl (1 reaction volume) aliquots of the SH gene
    master mix into wells of the appropriate columns of a MicroAmp
    optical 96-well reaction plate per the sample plate worksheet.

7.  For each sample, add 2.5 µls of RNA into two consecutive wells per
    the sample plate worksheet. (Total volume in each well should now be
    25 µls.)

8.  The following standards and controls should be run on each plate per
    the sample plate worksheet.

    a.  Mumps infected cell RNA standard:

        i.  Make serial ten-fold dilutions of RNA (Genotype D, 118 ng/ul
            starting concentration)

        ii. Add 2.5 µl from each 10^-5^, 10^-4^, 10^-3^, and 10^-2^
            dilution into two wells each.

    b.  Include nuclease-free water (2.5 µls) as a non-template control
        (NTC) for all master mix preparations.

9.  Seal the plate with an optical adhesive cover and centrifuge at 1500
    rpm for 3 minutes at room temperature. Wrap the plate in foil and
    hold at 4^o^C until ready to run.

10. Before running on the ABI Prism 7900HT Sequence Detection System,
    place a compression pad (Applied Biosytems #4312639) on top of the
    sealed reaction plate.

    a.  Thermocycling parameters:

> RT Step: 48^o^C/30 minutes
>
> AmpliTaq Gold Activation: 5^o^C/5 minutes
>
> PCR (40 cycles) 95^o^C/15 seconds
>
> 60^o^C/1 minute

The following procedure has been show to produce comparable results to
the one listed above.

+---------------------+---------+--------+----------+------+---------+
| Component           | Lot \#  | Volum  | \        | \#   | Total   |
|                     | and     | e/tube | [Final\] | R    | volume  |
|                     |         | (µl)   |          | xns. | (µl)    |
|                     | Exp.    |        |          |      |         |
|                     | Date    |        |          |      |         |
+---------------------+---------+--------+----------+------+---------+
| RNase-free H~2~O    |         | 9.25   |  -       |      |         |
+---------------------+---------+--------+----------+------+---------+
| 10X TaqMan buffer   |         | 2.5    | 1X       |      |         |
+---------------------+---------+--------+----------+------+---------+
| 25 mM magnesium     |         | 5.5    | 5.5 mM   |      |         |
| chloride            |         |        |          |      |         |
+---------------------+---------+--------+----------+------+---------+
| 10 mM dATP          |         | 0.75   | 300 µM   |      |         |
+---------------------+---------+--------+----------+------+---------+
| 10 mM dCTP          |         | 0.75   | 300 µM   |      |         |
+---------------------+---------+--------+----------+------+---------+
| 10 mM dGTP          |         | 0.75   | 300 µM   |      |         |
+---------------------+---------+--------+----------+------+---------+
| 20mM dUTP           |         | 0.75   | 600 µM   |      |         |
+---------------------+---------+--------+----------+------+---------+
| Forward primer      |         |  0.5   | (see     |      |         |
|                     |         |        | below)   |      |         |
+---------------------+---------+--------+----------+------+---------+
| Reverse primer      |         |  0.5   | (see     |      |         |
|                     |         |        | below)   |      |         |
+---------------------+---------+--------+----------+------+---------+
| Probe               |         |  0.5   | (see     |      |         |
|                     |         |        | below)   |      |         |
+---------------------+---------+--------+----------+------+---------+
| AmpliTaq Gold (5    |         | 0.125  | 0.025    |      |         |
| U/µl)               |         |        | U/µl     |      |         |
+---------------------+---------+--------+----------+------+---------+
| MultiScribe RT (50  |         | 0.125  | 0.25     |      |         |
| U/µl)               |         |        | U/µl     |      |         |
+---------------------+---------+--------+----------+------+---------+
| RNase inhibitor     |         | 0.5    | 0.4 U/µl |      |         |
+---------------------+---------+--------+----------+------+---------+
| RNA                 |         | 2.5    |  -       |      |         |
+---------------------+---------+--------+----------+------+---------+
| Total               |         | 25     |          |      |         |
+---------------------+---------+--------+----------+------+---------+

Comments, suggestions and questions concerning this procedure may be
sent to:

CDC: Paul Rota \"Rota, Paul \\(CDC/NCID/VR\\)\"
\<[par1@CDC.GOV]{.underline}\>

UHL: Lucy DesJardin \"Lucy DesJardin\"
\<[ldesjard@uhl.uiowa.edu]{.underline}\>