Lepu Medical Lepgen-96 Technical manual

Real-Time PCR System
Model: Lepgen-96
Operation Instruction
BEIJING LEPUMEDICAL TECHNOLOGY CO., LTD.

Contents
1. Introduction to the Real-Time PCR System.................................................................................................... 2
1.1 System description......................................................................................................................................2
1.1.1 Product overview .....................................................................................................................2
1.1.2 Product performance................................................................................................................2
1.2 Scope of application....................................................................................................................................5
1.3 Applicable population.................................................................................................................................5
1.4 Main components........................................................................................................................................5
1.5 Product packaging list.................................................................................................................................5
1.6 Panel introduction.......................................................................................................................................5
1.6.1 Introduction to front panel .......................................................................................................5
1.6.2 Introduction to sample pool .....................................................................................................6
1.6.3 Introduction to rear panel.........................................................................................................6
1.7 Working, transportation and storage conditions of Real-Time PCR System ..............................................7
2. Installation of the Real-Time PCR System.......................................................................................................7
2.1 Installation Environment Requirements......................................................................................................7
2.2 System installation......................................................................................................................................8
2.2.1 Installation of power cables .....................................................................................................8
2.2.2 Software installation ................................................................................................................8
2.2.3 Drive installation....................................................................................................................10
2.2.4 Software uninstallation ..........................................................................................................12
3. Introduction to the software operation of the Real-Time PCR System .......................................................12
3.1 Creating experiments ................................................................................................................................13
3.1.1 Experimental design...............................................................................................................14
3.1.1.1 Experimental information ...........................................................................................14
3.1.1.2 Plate well design .........................................................................................................14
3.1.1.3 Sample list...................................................................................................................15
3.1.1.4 Heating program .........................................................................................................16
3.1.2 Experiment run.......................................................................................................................17
3.1.3 Result analysis .......................................................................................................................18
3.1.3.1 View and analyze the experimental results .................................................................19
3.1.3.2 Export and storage of experimental data.....................................................................20
3.1.4 Experiment report ..................................................................................................................20
3.1.5 Open recent experiments........................................................................................................21
3.1.6 Open recent template .............................................................................................................21
3.2 Item management......................................................................................................................................21
3.2.1 Item settings...........................................................................................................................22
3.2.2 Result determination settings .................................................................................................23
3.3 Dye management ......................................................................................................................................23
3.4 Software application .................................................................................................................................24
3.4.1 Absolute quantitative / qualitative experiment.......................................................................24
3.4.1.1 Absolute quantitative / qualitative experiment process...............................................25
3.4.1.2 Absolute quantitative / qualitative experimental analysis ...........................................27
3.4.2 Melting curve experiment ......................................................................................................27
3.4.2.1 Standard melting curve experimental process.............................................................28

Operation Instruction for Real-Time PCR System (Model: Lepgen-96)
3.4.2.2 Analysis of melting curve ...........................................................................................29
4. Warnings, Precautions and Contraindications...............................................................................................29
4.1 Operation precautions...............................................................................................................................29
4.2 Power voltage, connection and grounding................................................................................................29
4.3 Applied process of the Real-Time PCR System........................................................................................30
4.4 Contraindications......................................................................................................................................30
5. Fault Phenomenon and Troubleshooting ........................................................................................................30
6. Maintenance, Repair and Cleaning.................................................................................................................32
6.1 Maintenance, repair and calibration..........................................................................................................32
6.1.1 Maintenance and repair..........................................................................................................32
6.1.2 Calibration .............................................................................................................................32
6.2 Cleaning and maintenance of Real-Time PCR System.............................................................................32
6.3 Fuse replacement ......................................................................................................................................33
6.4 Other precautions......................................................................................................................................33
7. Interpretation of Graphics, Symbols and Abbreviations on Labels of Medical Devices ............................33
8. Electro Magnetic Compatibility (EMC) Description.....................................................................................34
8.1 Electromagnetic radiation .........................................................................................................................34
8.2 Electromagnetic interference ....................................................................................................................35
8.3 Calculation of spacing between communication device and system ........................................................36
8.4 Spacing between system and communication device ...............................................................................37
9. Electrical Safety ................................................................................................................................................38
10. Service Life......................................................................................................................................................38
11. Manufacturer Information.............................................................................................................................38

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 1
Introduction to the Instruction for Use
The Instruction for Use of the Real-Time PCR System consists of the following parts:
Introduction: general introduction to the Real-Time PCR System;
•Installation operation: guide the user to install all parts of the Real-Time PCR System;
Testing and setting: guide the user to test and set up the Real-Time PCR System;
Warning, failure and maintenance: guide the maintenance of the Real-Time PCR System, handle common failures,
as well as warning labels and manufacturer information.
Pay attention to the precautions under this mark.

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 2
1. Introduction to the Real-Time PCR System
1.1 System description
1.1.1 Product overview
The Real-Time PCR System is used for real-time PCR assay and analysis of experimental data. With the help of
corresponding reagents, this instrument can make the target nucleotides of objects to be detected amplified and
fused under specific temperature conditions which are reflected in the form of fluorescence, and judge the target
nucleotides qualitatively and quantitatively or study the melting curve and genetic typing of the target
nucleotides by analyzing the fluorescence signals generated in the experiment.
The working principle of the Real-Time PCR System is: after the probe labeled with fluorophore is mixed with
the unknown DNA template, the thermal cycle of high temperature denaturation, low temperature renaturation
and thermophilic extension is completed; according to the polymerase chain reaction (PCR) law, the probe
complementary to the unknown DNA template is cut off, the fluorophore is free in the reaction system and emits
fluorescence under specific light excitation; with the increase in cycle times, the target gene fragment amplified
increases exponentially, and the fluorescence intensity of the reaction system increases accordingly. The copy
number of the target gene of the sample to be tested can be obtained by obtaining the Ct value through real-time
detection of the corresponding fluorescence signal intensity and by quantitatively analyzing the unknown DNA
template based on the standard curve of several standards with known DNA template concentration.
The instrument is mainly used for qualitative or quantitative analysis on a variety of genes in the clinical
laboratory by means of fluorescence PCR.
1.1.2 Product performance
Temperature control
1) Heating rate
Average heating rate: from 48 ℃ ~ 90 ℃, no less than 1.5℃/s.
2) Cooling rate
Average cooling rate: from 90℃ ~ 50℃, no less than 1.5℃/s.
3) Module temperature control accuracy
It should not be more than 0.5℃.
4) Temperature accuracy
The absolute value of the difference between the measured value and the set temperature shall not be greater
than 0.5℃.
5) Module temperature uniformity
The temperature difference should be within ± 1℃.
6) Temperature duration accuracy
The relative deviation between the temperature duration and the programmed temperature duration is within ± 5%.
Fluorescence intensity detection
1) Repeatability of fluorescence intensity detection
The coefficient of variation (CV,%) should not be greater than 3% after repeated detection with each calibration dye
with high, medium and low concentrations.
2) Precision of fluorescence intensity detection
Within the range of instrument measurement, 10 test wells are randomly selected, and each calibration dye of high,
medium and low concentrations is used for detection and the coefficient of variation (CV,%) should not be greater
than 5%.
3) Fluorescence interference in different channels

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 3
The cross interference from other channels is less than 1.00%.
Sample test repeatability
For testing of nucleic acid samples with high, medium and low concentrations, the CV of Ct value (or log value of
concentration) should not be greater than 3%.
Linearity
1) Sample linearity
The samples with the concentration of series dilution gradients (5 gradients) are tested. The Ct value of each
concentration and the absolute value of linear regression coefficient r of the log value of concentration are not less
than 0.980.
2) Fluorescence linearity
The samples of fluorescent dye with the concentration of series dilution gradients (5 gradients) are tested. The
measured value of fluorescence with each concentration and the absolute value of linear regression coefficient r
under a dilution rate are not less than 0.990.
Appearance
1) The graphic symbols and texts on the panel should be accurate, clear, uniform and free of scratches;
2) The fastener connection should be firm and reliable without looseness;
3) The moving parts should be stable, free from jamming, kicking and significant backlash, and the key group
should be flexible.
General requirements for software
1. Object processing
Real-time fluorescence PCR data.
2. Maximum concurrency
Number of maximum concurrent user: 1.
3. Data interface
The data interface of the Real-Time PCR System is RS232 serial port.
4. Specific software and hardware
Windows Office Word / Excel 2007-2016 version.
5. Restrictions on use
1) The range of sample reaction system is 15-100 μ L;
2) The temperature setting range of the experimental procedure is 4.0-99.0 degrees;
3) The temperature duration is from 00:00 to 99:59;
4) The maximum number of run segments of the experimental procedure is 9, and each run segment covers 9 steps
at most;
5) A maximum of 99 cycles can be set in each run segment;
6) Only one step can be set as fluorescence detection step in each run segment, and the temperature duration of this
step must be greater than 15s, and the sum of cycle times of all run segments must be greater than 3;
6) Baseline start range: 1 ~ (baseline end-1); baseline end range: (baseline start+1) ~ the sum of cycle times of all
run segments set.
6. User access control
Users can directly access the software without ID authentication.
7. Copyright protection

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 4
The software is sold with the equipment in the form of CD, and the serial number provided shall be used for the
installation of the software. The software can be used indefinitely after installation, without restrictions on the
number and time of use.
8. User interface
Menu interface: icon menu, text and icon mixed menu;
Dialog interface: answer type, warning type;
Data input interface;
Data display interface.
9. Message
The message types of the software are as follows:
General prompt message: prompt message after you enter data to proceed to the next, deletion prompt, file change
saving prompt when closing, and exit prompt.
Error prompt message: communication error prompt, prompt of hot cover not pushed in place.
Device status message: in the process of running the experiment, the current real-time temperature, real-time
fluorescence data, current running status and running time will be displayed in real time.
10 Reliability
1) The software protects the reliability against user input boundary value and semantic error.
The software protects the recoverability against user's abnormal operation errors:
2) During the experiment running, if the software is forced to shut down (process interruption or computer power
failure), the user can choose whether to continue the operation after restarting the software.
During the experiment running, if the serial port line is unplugged and the communication is reconnected, the user
can choose whether to continue the operation.
3) Users' data can be exported for backup at any time.
11. Maintainability
The PCR upper computer provides maintenance information in the form of log. The log file is in the log folder of
the root directory of the software. The name of the log file is Logxxxxxxxx.txt (XXXXXXXX is the date when the
log is generated; for example, Log 20160817.txt, which represents the log information on August 17, 2016).
The log content includes the following information: experimental record call information (including the name and
call time of experimental record), time of new experiment, time of experiment start and end, communication and
device exception information (including communication exception information).
12. Efficiency
During the experiment running, the receiving time interval of real-time temperature data and fluorescence signal
data (such as detecting fluorescence) is not more than 150 ms.
The time from communication disconnection to error prompt shall not exceed 18 s.
13 Operating environment
1) Hardware configuration
Processor: Intel or AMD dual core 2.8 GHz
Memory: more than 4 G
Hard disk capacity: more than 128 GB
Peripheral port: USB port
2) Software environment:
System software: Windows 7 (32-bit)/8 (32-bit)/8.1 (32-bit).

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 5
Quality requirement
According to the requirements of Chapter 5 of GB/T 25000.51.
1.2 Scope of application
Based on the principle of polymerase chain reaction (PCR) and real-time fluorescence monitoring technology, the
product is used together with supporting detection reagents to carry out qualitative and quantitative detection or
melting curve detection of analytes in nucleic acid samples (DNA/RNA) from human body, including nucleic acid
of pathogens and human gene.
1.3 Applicable population
Target patient population: adults, children or newborns that clinicians think need real-time fluorescence PCR
detection.
Patient selection criteria: patients need to provide enough clinical samples to meet the requirements of the test.
1.4 Main components
This product is composed of a control module, a power module, a temperature control module, an optical path
detection module, a shell module, a hot cover module, a support module, as well as upper computer software and
embedded software specific to the Real-Time PCR System.
1.5 Product packaging list
Product packaging list
Components
Quantity
Real-Time PCR System (model: Lepgen-96)
1
Power cord
1
USB serial port line (USB-9 pin)
1
Warranty card
1
Operation Instruction
1
Balloon (optional)
1
Software installation CD
1
Fuse (Φ 5 * 20 mm, 5 A, 250 V)
2
Certificate
1
Packing list
1
1.6 Panel introduction
1.6.1 Introduction to front panel
The front panel is illustrated as follows:

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 6
Fig. 1-1 Front Panel
As shown in Fig. 1-1, when the instrument is turned on, the power indicator will be on; when the experiment
running is programmed, the running indicator light will be on and off when the program running is finished; each
time when the fluorescence detection is carried out, the detection indicator light will be on and off when the
fluorescence detection is finished. During the whole process of program run, the running indicator will be always
on and the detection indicator will be on and off with the detection stage.
1.6.2 Introduction to sample pool
Fig. 1-2 Sample Pool
As shown in Fig. 1-2, the instrument adopts constant temperature bath and fluorescence detection. There are 96
test tube wells in the sample pool. Each well will undergo corresponding heating, temperature holding and
cooling process with the running of the temperature program edited by the user. Different channels in each well
are tested according to the temperature program edited. Note that at the end of the experiment, the temperature
of the reaction pool may be high, and the user should pay attention to burn prevention when taking samples.
1.6.3 Introduction to rear panel
The rear panel includes a power socket, a switch, a fuse, COM1 port and COM2 port.
The power socket, switch and fuse are integrated, which are used to turn on and off the power supply of the device.
Running indicator

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 7
Fig. 1-3 Rear Panel
1.7 Working, transportation and storage conditions of Real-Time PCR System
Specification
Input voltage
AC220～,50Hz
Input power
800VA;
Dimensions
490mm x355mm x284mm;
Weight
20Kg;
Working conditions
1) Temperature: 10～30℃
2) Relative humidity: < 85%;
3) Atmospheric pressure: 78.9 kPa ～ 101.3 kPa
Storage conditions
Storage temperature: - 20℃ ~ 55℃, relative humidity: ≤ 90%, no corrosive gas,
and well-ventilated.
Transport conditions
Transportation temperature: +10℃ ~+40℃, relative humidity: 30% ~ 75%.
During transportation, it shall be protected from severe impact, rain and sun
exposure.
2. Installation of the Real-Time PCR System
2.1 Installation Environment Requirements
◆ Hardware environment requirements:
1) The host of PCR device must be placed on a horizontal and secure working platform, and be away from direct
sunlight, heating equipment and dust;
2) The device host should be kept at least 12 cm away from the wall or surrounding objects to ensure the normal
ventilation and heat dissipation of the device, and to facilitate the user to turn on and off the power supply of the
instrument or connect other equipment such as computers;
3) The device shall be kept far away from instruments with strong electromagnetic interference or high induction
coefficient, such as high-speed centrifuge, oscillator, etc.
◆ Software environment requirements:
1) Hardware configuration
Processor: Intel or AMD dual core 2.8 GHz

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 8
Memory: more than 4 GB
Hard disk capacity: more than 128 GB
Peripheral port: USB port
Note: if the hardware configuration cannot meet the above requirements, the software operation efficiency and user
experience will be affected.
2) Software environment:
System software: Windows 7/8/8.1/10
Specific software: Windows Office Word / Excel 2007-2016 version.
2.2 System installation
2.2.1 Installation of power cables
1) Take out the supporting power cable from the packing box;
2) Insert one end of the power cable into the external power supply with grounding protection;
3) Insert the other end of the power cable into the power interface behind the Real-Time PCR System;
4) During the installation, ensure that the power switch of the Real-Time PCR System is disconnected.
2.2.2 Software installation
1) Read the software installation CD and open the software installation package in the CD of the Real-Time PCR
System.
2) Double click the software installation package to open the installation dialog box, as shown in the following
figure:
Fig. 2-1
3) Click “OK”
Fig. 2-2

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 9
4) Click “Next”
Fig. 2-3
4) Click “Install” to open the following installation interface, and wait for the installation to complete.
Fig. 2-4
4) After installation, click “Finish” to automatically open the Real-Time PCR System software.
Fig. 2-5

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 10
2.2.3 Drive installation
1) First, do not connect the serial port line to the computer
2) Double click the Real-Time PCR System software installation package\Drivers\CDMInstaller file, and the
following window will pop up:
Fig. 2-6
3) Click “Extract” to open the following interface:
Fig. 2-7
4) Click “Next”

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 11
Fig. 2-8
5) Click “I accept this agreement”, and then click “Next”, the driver installation interface will appear,
Fig. 2-9
6) After installation, click “Finish”
Fig. 2-10

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 12
2.2.4 Software uninstallation
1) Close the Real-Time PCR System software
1) Click Windows “Start” menu - > All Programs - > LepuPCR - > uninstall LepuPCR, as shown below:
Fig. 2-11
3) In the pop-up window, select “Yes”, as shown below:
Fig. 2-12
4) Wait until the uninstallation is finished, as shown below:
Fig. 2-13
3. Introduction to the software operation of the Real-Time PCR System
Connect the power cable of the Real-Time PCR System to the 220V socket, close the power switch, open the
Real-Time PCR System, enter the Windows system interface after the equipment is powered on, and double
click the LepuPCR icon on the desktop to test. The main interface of the software consists of Menu Bar,
Toolbar, Experiment Tab, Recent Experiment and Recent Template, as shown in the following figure:

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 13
Fig. 3-1 Main Interface
3.1 Creating experiments
Mix according to the instructions of the reagent, put the prepared solution sample into the reagent tube, and close
the cap of the reagent bottle. The sample capacity in the reagent tube is required to be more than 15 uL. Put the
sample into the sample pool of the instrument.
The software can create experiments in two ways. The first is to click “Experiment” in the menu bar of the main
interface, and the second is to click “New Experiment” in the toolbar of the main interface, as shown in Fig. 3-2
and Fig. 3-3:
Fig. 3-2
Fig. 3-3
Click any experiment type, to automatically jump to the new experiment interface, which guides the user to
carry out experiment design in a flow-based way.

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 14
3.1.1 Experiment Design
The experimental design consists of experiment Info, Well Design, Sample Table and Thermal Profile, as shown
in the following figure:
Fig. 3-4 Experimental Design
3.1.1.1 Experiment Info
The experiment info interface is mainly used to edit and display the basic experiment information and instrument
setting information, as shown in the following figure:
Fig. 3-7 Experimental Information
Experiment Name: defaulted as the time when the experiment is created (the default format is
year-month-day_hour-minute-second). Users can click here to edit as needed.
Experiment type: you can select the appropriate experiment type from the pull-down menu, such as "Absolute
Quantitative/Qualitative Experiment" and "Melting curve Experiment".
Algorithm setting: can be set as relative fluorescence value method and absolute fluorescence value method.
Experiment setting: set the sample size, whether to use the heat cover or not, and set the temperature control mode.
Data setting: set whether the patient information is saved in the database.
3.1.1.2 Well Design
Click the Well Design tab to enter the well design interface. Well Design is mainly to edit and display the item
information, patient information, well information and channel settings of reaction well, as shown in Fig. 3-5:

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 15
Fig. 3-5 Well Design
1) Select the reaction well of corresponding reagent in the reaction well plate, and the background of the selected
reaction well becomes dark gray.
You can click to select 1 reaction well on the reaction well plate, or press and hold Ctrl key, and click multiple
reaction wells at the same time to select multiple discontinuous reaction wells. You can also drag the mouse to
select the corresponding area on the reaction well plate and press the Direction Key to move up, down, left and
right.
2) In the Project Info tab, select the project you want to test.
The Project Info tab displays the item information that the user saved in the software. If the item information
required by the user is not available in the item list, it is necessary to contact the technical service personnel of the
reagent manufacturer or create the item by yourself according to the Instruction 3.2.
3) In the patient info tab, enter patient number, name, gender, age and other information.
4) In the well info tab, users can modify the well type, sample number, and sample type.
3.1.1.3 Sample Table
Click the “Sample Table” tab to enter the sample table interface, which is used to display all the information of the
current samples, as shown in the following figure:

Operation Instruction for Real-Time PCR System (Model: Lepgen-96) 16
Fig. 3-8 Sample List
3.1.1.4 Thermal Profile
Click the “Thermal Profile” tab to enter the thermal profile setting interface, as shown below.
Fig. 3-6 Thermal Profile
The Thermal Profile can be imported and exported.
Common editing of thermal profile
Click New Running Stage (before) , you may add ordinary, constant temperature,
amplification and other program segments before the program segment selected; click New Running Stage (after)
, you may add them before the program segment selected.
Click New Step , you can add steps before and after the step selected; click Delete
, you can be delete the step.