Sansure Biotech

Mycoplasma Pneumoniae DNA Fluorescence Diagnostic Kit

(PCR-Fluorescence Probing)

【 Reference Number 】
S3016E
【 Product Name 】
Mycoplasma Pneumoniae DNA Fluorescence Diagnostic Kit (PCR-Fluorescence Probing)
【 Package Specification 】
48 tests/kit
【 Intended Use 】
Mycoplasma pneumoniae (MP) is a pathogenic microorganism between bacteria and virus. It is mainly transmitted
through buccal and nasal mucus by the air causing respiratory diseases, with the highest incidence in children and
adolescents. Respiratory infection has the manifestations of pharyngitis and bronchitis, with a few cases causing infection
to the lung. Recently, incidence among infants and children is increasing, therefore, early diagnosis and treatment can
decrease the exacerbation of acute pneumonia in children. The development of molecular biology also draws more
attention to the fluorescence quantitative PCR technology for the detection of MP-DNA.
This diagnostic kit is an in vitro nucleic acid amplification test for the detection of mycoplasma pneumoniae DNA in human
sputum and throat swab. It is intended for use as an aid in the diagnosis of an MP infection and providing a
molecular-diagnostics-based solution.
【 Test Principle 】
The diagnostic kit uses a nucleic acid lysis buffer to allow rapid lysis and release of MP-DNA from a sputum specimen or
throat swab which has been processed with concentrate. By applying real-time fluorescence PCR technology, this test
uses a pair of specific primers which are designed to target a conserved sequence of MP-DNA, and a specific
fluorescence probe, accompanied with other components in PCR mix, to achieve quick detection of MP-DNA through
fluorescent signal changes.
The PCR detection system uses UNG enzyme + dUTP contamination-proof system, which can fully degrade possible
unwanted side-products, to avoid a false positive result.
The PCR detection system uses internal control to monitor the presence of PCR inhibitors in order to avoid a false
negative result.
【 Components of the Diagnostic Kit 】
No. Reagent Name Specification & Qty. Main Ingredients
1 MP-Lysis Buffer 2.5 mL/tube × 1 tube KCl, SDS, Surfactin
2 MP-Enzyme Mix 96 μL/tube × 1 tube DNA polymerase, UNG enzyme
3 MP-Internal Control 50 μL/tube × 1 tube Cloning plasmid containing the target gene fragment
4 MP-PCR Mix 912 μL/tube × 2 tubes Primer, probe, dNTPs, Mg2+, PCR buffer solution
5 MP-Positive Reference A
50 μL/tube × 1 tube Cloning plasmid containing the target gene fragment
(4.00E+07 copies/mL)
6 MP-Positive Reference B
50 μL/tube × 1 tube Cloning plasmid containing the target gene fragment
(4.00E+06 copies/mL)
7 MP-Positive Reference C
50 μL/tube × 1 tube Cloning plasmid containing the target gene fragment
(4.00E+05 copies/mL)
8 MP-Positive Reference D
50 μL/tube × 1 tube Cloning plasmid containing the target gene fragment
(4.00E+04 copies/mL)
9 MP-Negative Control 50 μL/tube × 1 tube MP negative specimen (inactivated)
10 MP-Positive Control 50 μL/tube × 1 tube MP positive specimen (inactivated)

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11 Concentrate 5 mL/tube × 1 tube PEG-6000, NaCl, purified water
Note: 1. Do not mix or interchange components from different kit lots.
2. All biological specimens in the diagnostic kit have been inactivated.
3. Self-prepared reagent: sterile saline.
4. Specimen processing: it is recommended to use Sansure Biotech’s magnetic-bead DNA extraction reagent to extract
DNA, then there is no need to process the DNA by lysis buffer.
【 Storage 】
The reagents should be stored in sealed bottles at -20±5°C and protected from light. The shelf life of the diagnostic kit is
12 months. Multiple freeze/thaw cycles should be avoided.
【 Compatible Instrument 】
The diagnostic kit is compatible to ABI7500, Mx3000P and Roche LightCycler 480.
【 Specimen Requirements 】
1. Applicable specimen type: sputum and throat swab.
2. Collection of specimen
2.1 Collection of sputum: place sick children in a horizontal position with the head up. Use single-use sputum aspirator.
Put the aspirator's tube slowly and gently in the throat and adjust it to negative-pressure to aspirate steadily the secretion
laying in the deep of patients' airways(It is recommended to aspirate atomized sputum). Repeat the aspiration several
times. Use the collected sputum as specimen and seal it and send it for detection. The collected sputum should be sent for
detection as soon as possible before it goes dry and becomes invalid for detection.
2.2 Collection of throat swab: use sterile cotton swab to collect secretions in the throat and place the throat swab in a
sterile glass tube. Seal it and send it for detection.
3. Storage and delivery of specimens:
Specimens collected via the above-mentioned method can be used for immediate detection, or stored at 2-8°C for up to
24 hours, or below -20°C for a long term storage. Multiple freeze/thaw cycles should be avoided. Specimens should be
transported in a sealed frozen pitcher with ice or in a sealed foam box with ice.
【 Test Method 】
1. Preparation of reagent (performed at “reagent preparation region”)
1.1 Take out each component from the diagnostic kit and place them at room temperature. Allow the reagents to
equilibrate at room temperature, and then vortex each of them respectively for future use.
1.2 According to quantity of test specimens, MP-negative control and MP-positive control, pipette appropriate quantity of
MP-PCR mix, MP-enzyme mix and MP-internal control (MP-PCR mix 38 μL/test+ MP-enzyme mix 2 μL/test+ MP-internal
control 0.4 μL/test), fully mix them to make a PCR-Mastermix and centrifuge it instantaneously for future use.
1 sample 10 samples 24 samples 48 samples
MP-PCR mix (μL) 38 380 912 1824
MP-enzyme mix (μL) 2 20 48 96
MP-internal control (μL) 0.4 4 9.6 19.2
Note: The above configuration is just for your reference and to ensure enough volume of
the PCR-Mastermix, more volume of the actual pipetting may be required.
2. Processing and loading of specimens (performed at “specimen processing region”)
2.1 Processing of MP-negative control, MP-positive control and MP-positive references
Pipette 10 μL of MP-negative control, MP-positive control and MP-positive references A-D respectively and then mix each
of them with 10 μL of lysis buffer respectively for future use.
2.2 Processing of sputum
Add the normal saline into sputum specimen with the volume of saline two to three times to specimen. Vortex it thoroughly
and hold it to liquefy the sputum. Pipette 1000 μL of liquefied specimen to a 1.5 mL centrifuge tube (be careful not to
pipette solid impurities out). Add 100 μL of concentrate to the centrifuge tube. Vortex it and thoroughly mix it. Centrifuge it

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at 12,000 rpm for 5 minutes. Discard the supernatant (It is recommended to leave 20 μL of the supernatant in the tube ).
Add 50 μL of MP-lysis buffer into the precipitate and vortex it or pipette it up and down to mix it and then hold it for 10
minutes to allow thorough lysis for future use.
2.3 Processing of throat swab
Add 1 mL of sterile saline into specimen collection tube. Vortex it and mix it thoroughly. Then transfer all the liquids
(specimen eluent) to a 1.5 mL centrifuge tube (press to dry the cotton swab against the centrifuge tube wall and then
discard the swab). Add 100 μL of concentrate to the centrifuge tube. Vortex it and thoroughly mix it. Centrifuge it at 12,000
rpm for 5 minutes. Discard the supernatant (it is recommended to leave 20 μL of supernatant in the tube). Add 50 μL of
MP-lysis buffer into the precipitate and vortex it or pipette it up and down to mix it and then hold it for 10 minutes to allow
thorough lysis for future use.
2.4 Loading of specimens
2.4.1 Add 10 μL of the above processed test specimen, MP-negative control, MP-positive control and MP-positive
references A-D respectively into each PCR reaction tube.
2.4.2 Add 40 μL of PCR-Mastermix into each tube. Pipette it up and down for 2-3 times. Cover the tube lid (Remove the
bubbles). Centrifuge it at 2000 rpm for 30 seconds.
3. PCR Amplification (performed at “amplification and analysis region”) (refer to user manual of each instrument to adjust
the settings)
3.1 Place the PCR reaction tube into the specimen well of the amplification device. Set up the MP-negative control,
MP-positive control, MP-positive references A-D and unknown samples in the corresponding sequence and input sample
information and concentration of MP-positive references A-D.
4.2 Select PCR test channel
3.2.1 For ABI, Stratagene series:
a. Select FAM channel (Reporter: FAM, Quencher: None) to test MP-DNA.
b. Select HEX or VIC channel (Reporter: HEX/VIC, Quencher: None) to test MP-internal control.
c. Set passive reference: none.
d. Set sample volume: 50.
3.2.2 For Roche LightCycler 480:
Choose “New Experiment”. Click “Dual Color Hydrolysis Probe/ UPL Probe” in the drop-down menu of setup
panel-Detection format. Do the following in the drop-down menu of “Customize”:
a. Select FAM channel to test MP-DNA;
b. Select VIC/HEX/Yellow 555 channel to test internal control.
c. Set reaction volume: 50.
3.3 Set cycle parameters (the time parameter varies according to instruments):
3.3.1 ABI, Stratagene series:
Step Temperature Time Cycle No.
1 UNG enzyme reaction 50°C 2 min. 1
2 Taq enzyme activation 94°C 5 min. 1
Denaturation 94°C 15 sec.
3 45
Annealing, extension, fluorescence collection 57°C 30 sec.*
4 Device cooling(optional) 25°C 10 sec. 1
Note: Due to the device ABI 7500’s technical specification, it can not be set at 30 sec. but 31 or 32 sec.)
When the setting is completed, save settings and carry out the reaction procedure.
3.3.2 LightCycler 480 (choose default value for non-listed parameters)
Program Target(°C) Acquisition Mode Hold (hh:mm:ss) Cycles Analysis Mode
1 50 None 00:02:00 1 None
2 94 None 00:02:00 1 None

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94 None 00:00:05
3 45 Quantification
57 Single 00:00:30
4 (optional) 25 None 00:00:10 1 None
When the settings are completed, save settings, operate the reaction procedure.
4. Result Analysis (Refer to user manual of each instrument to adjust the settings)
When the reactions are completed, results will be saved automatically. After analysis, adjust Start, End and Threshold
values of Baseline of the graph (users can adjust them according to the actual situations. Start value can be set at 3-15,
end value at 5-20. Adjust the amplification curve of negative control to be flat or below threshold). Click “Analyze” to
implement the analysis and make sure each parameter satisfies the requirements given in “5. Quality Control”. Go to
“Plate” window to record the Ct value.
5. Quality Control
The test result is treated as valid if all the conditions in the table below are met for the same test. Otherwise the test result
is treated as invalid and needs to be re-tested.
MP-Positive Control MP-Negative Control MP-Internal Control MP-Positive References
(A, B, C, D)
Ct value ≤ 30 N/A ≤ 40 ≤ 39
【 Reference Range 】
Through the research on reference values, the Ct reference value of target gene is determined to be 39, and the Ct
reference value of internal control is determined to be 40.
【 Explanation of Detection Result 】
Conclusion Ct value of Ct value of internal Amplification curve of
sample control sample
Positive ≤ 39 - like "S"
Negative N/A ≤ 40 like "--"
Out of limit (concentration < 4.00E+02 >39 ≤ 40 like "S"
copies/mL)
Invalid* - > 40 or N/A -
Note: *This suggests an investigation should be carried out to find out the reasons when Ct value of the internal control
is > 40 or N/A and retest it. (If repeated tests still produce invalid results, please contact Sansure Biotech at
info@sansure.com.cn)
【 Limitations of Detection Method 】
Detection result is related to specimen collection, processing, delivery and storage quality. Any deviation from the stated
procedure will lead to an inaccurate detection result. Cross-contamination during specimen processing may also result in
a false-positive result.
【 Product Performance Index 】
When the kit is used to detect the enterprise’s work references, the consistency rate for both negative and positive
reaches 100%. Precision test shows excellent reproducibility in both intra-batch and inter-batches with its coefficient of
variation of Ct value <10%, and its coefficient of variation of concentration <50%. The sensitivity of this kit is determined to
be 4.00E+02 copies/mL. It shows no cross-reaction with pathogens such as UU, CP, TB, EBV and influenza virus.
【 Precautions 】
1. The product can only be used for in vitro diagnosis. Please read the product manual carefully before operation.
2. Please learn and be familiar with the operation procedures and precautions for each instrument before test. Please
make sure quality control for each test.
3. Laboratory management shall strictly follow management practices of PCR gene amplification laboratory; laboratory
personnel must receive professional training; test processes must be performed in separated regions; all consumables
should be for single use only after sterilization; special instruments and devices should be used for every process; all lab

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devices used in different processes and regions should not be cross-used.
4. All specimens for detection should be handled as if infectious. Wear laboratory coats, protective disposable gloves and
change the gloves often to avoid cross-contamination between samples. Handling of specimens and waste must meet
relevant requirements outlined in local, state and national regulations.
5. Before use, all reagents must be fully thawed at room temperature and mixed thoroughly.
6. If there is no HEX or VIC test channel for fluorescence PCR instrument, monitoring of internal control can be omitted.
D0 not add internal control at the above step of 1.2 to avoid interference from extra multicolor fluorescence. Please
consult with Sansure staff about the option of detection channel.
【 Bibliography】
1. Sun Xiangyang, Yan linyi, Gao Xilan, Complications analysis of mycoplasma pneumoniae in 42 cases of children [J]
Modern Diagnosis and Treatment, 1999, 10(3): 45.
2. Hammerschlag M.R. Mycoplasma pneumoniae infections. Curr Opin Infect Dis 2001;14:181-6.
3. Daxboeck F., Krause R., Wenisch C. Laboratory diagnosis of Mycoplasma pneumoniae infection. Clin Microbiol Infect
2003;9:263-73.
4. Andreu L.M., Molinos A.S., Fernandez R.G., et al. Serologic diagnosis of Mycoplasma pneumoniae infections. Enferm
Infecc Microbiol Clin 2006;24 Suppl 1:19-23.
5. Jonas M. Winchell, Kathleen A. Thurman, Stephanie L. Mitchell. Evaluation of Three Real-Time PCR Assays for
Detection of Mycoplasma pneumoniae in an Outbreak Investigation. JOURNAL OF CLINICAL MICROBIOLOGY,
2008:Vol. 46, No. 9: 3116-3118.
【 Symbols 】

Symbols Meanings Symbols Meanings

In Vitro Diagnostic Medical Device Date of Manufacture

Use By Caution

Temperature Limitation Manufacturer

Authorized representative in the

Lot Number
European Community

Number of Tests Reference Number

Sansure Biotech Inc.

Add.: No. 680, Lusong Road, Yuelu District, 410205 Changsha, Hunan Province,
PEOPLE’S REPUBLIC OF CHINA
Tel.: +86-731-88883176
Fax: +86-731-88884876
Web: www.sansure.com.cn

Wellkang Ltd (www.CE-marking.com)

Add.: Suite B, 29 Harley Street, London W1G 9QR, UNITED KINGDOM

For Professional Use Only

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