Fluorescent quantitative PCR (qPCR) is the core equipment in the fields of molecular biology research, clinical diagnosis (such as detection of new coronavirus), and its accuracy directly affects the reliability of experimental results. Calibration is a key step in ensuring the stability of qPCR instrument performance, and it is necessary to regularly verify and adjust the temperature control system (temperature control accuracy) and optical system (fluorescence signal detection) systematically.

1、 The core objective of calibration

The calibration of qPCR instrument aims to ensure that the following key performance indicators meet the requirements:

Temperature control system: temperature indication error (deviation between set temperature and actual temperature), temperature uniformity (temperature difference between reaction holes), temperature fluctuation (temperature change over time), maximum temperature overshoot (amplitude exceeding the set value during heating/cooling process), average heating/cooling rate (heating/cooling speed).

Optical system: precision of fluorescence intensity (consistency of fluorescence signal in the same well), precision of threshold cycle number (Ct value) (consistency of Ct values in different wells), drift of melting temperature (Tm value) (temperature deviation when fluorescence intensity drops by half), linear correlation coefficient of fluorescence (linear relationship between fluorescence intensity and nucleic acid concentration).

2、 Preparation work before calibration

1. Environmental conditions

Temperature: 15-30 ℃ (to avoid temperature fluctuations affecting calibration results);

Relative humidity: 20-85% RH (to prevent the instrument from getting damp);

Power supply: Stable voltage (recommended to use UPS) to avoid power outages affecting the calibration process.

2. Equipment and materials

Standard substances: Certified standard substances (such as plasmid DNA and ribonucleic acid standard substances) must be used, and their characteristic values (copy number ≥ 10 copies/μ L) and relative expanded uncertainty (≤ 5%) must comply with the requirements of JJF (Tianjin) 04-2020;

Calibration equipment: optical simulator (integrated temperature sensor and emission light generator, temperature measurement range 0-120 ℃, maximum allowable error ± 0.1 ℃; emission light wavelength 360-780nm, adjustable relative light radiation intensity), multi-channel temperature calibrator (such as JDPC-96, used to measure temperature difference between reaction holes);

Consumables: specialized calibration plates (such as AB7500 ROI calibration plate, background calibration plate), pipettes (2 μ L, 10 μ L, etc., subject to metrological verification), powder free gloves (to prevent contamination of the calibration plate).

3、 The main process of calibration

The calibration process of qPCR instrument must strictly follow JJF (Tianjin) 04-2020 "Calibration Specification for Real time Fluorescence Quantitative PCR Instrument", mainly including temperature system calibration and optical system calibration. The following takes the AB7500 series instrument as an example to illustrate the specific steps:

（1） Temperature system calibration

Temperature is the core parameter of qPCR reaction, which directly affects the efficiency of DNA amplification. Temperature system calibration requires the use of an optical simulator (instead of a reaction tube) to simulate temperature changes in actual reactions and verify the temperature control performance of the instrument.

1. Preparation before calibration

Connect the optical simulator to the qPCR instrument, ensuring that the temperature sensor below is in good contact with the fluorescent emitting light source above and the instrument's heating block;

Apply thermal oil under an optical simulator to enhance heat conduction;

Open the instrument software, select the "Calibration" module, and enter the "Temperature Calibration" program.

2. Calibration Procedure

Preheating: Set the instrument temperature to 23 ℃ and run the preheating program (about 10 minutes) to bring the instrument to a stable state;

Temperature point setting: Select 9 temperature points (30 ℃, 50 ℃, 60 ℃, 70 ℃, 90 ℃, 95 ℃, etc.), each temperature point lasting for 2 minutes (used to measure temperature indication error, uniformity, fluctuation);

Heating/cooling process: from 50 ℃ to 90 ℃ (measured average heating rate), and then from 90 ℃ to 50 ℃ (measured average cooling rate);

Data collection: The instrument automatically records the set temperature (Ts), average actual temperature (T ₁), temperature fluctuation value (Tv), and maximum overshoot value (Tos) for each temperature point.

3. Result calculation

Temperature indication error: △ T=Ts - T ₁ (required to be ≤± 0.5 ℃);

Temperature uniformity: △ Tu=Tumax - Tumin (Tumax is the maximum temperature of all pores, Tumin is the minimum value, required to be ≤ 1.0 ℃);

Temperature fluctuation: △ Tv=± (Tmax - Tmin)/2 (Tmax is the maximum value of a single pore temperature, Tmin is the minimum value, and it is required to be ≤ 0.2 ℃);

Maximum overshoot at temperature: △ Tos=Tosmax - Ts (Tosmax is the maximum overshoot value for all holes, required to be ≤ 3.0 ℃);

Average heating rate: v_up=(TB - TA)/tot (TB is the average temperature point at 90 ℃, TA is the average temperature point at 50 ℃, tut is the heating time, required to be ≥ 4 ℃/s);

Average cooling rate: vwdown=(TB - TA)/tdo (tdo is the cooling time, required to be ≥ 3 ℃/second).

（2） Optical system calibration

The optical system is responsible for detecting fluorescence signals, and its accuracy directly affects the quantitative results of Ct values and nucleic acid concentrations. Optical system calibration requires the use of a dedicated calibration plate (such as AB7500 ROI calibration plate) to adjust the sensitivity and signal consistency of the fluorescence detector.

1. Background calibration

Purpose: To eliminate interference from instrument background fluorescence (such as electronic signals, plastic consumables contamination);

Steps:

(1) Remove the background calibration board (leave it at room temperature for 5 minutes to avoid condensation);

(2) Install the calibration board into the instrument and run the "Background Calibration" program (approximately 30 minutes);

(3) The software automatically records the background fluorescence signal and generates a background correction file (automatically deducted in subsequent experiments).

2. ROI (Region of Interest) calibration

Purpose: Map the well positions on the reaction plate to the instrument detector to ensure accurate collection of fluorescence signals from each well;

Steps:

(1) Remove the ROI calibration plate (standard plate or rapid plate, vortex for 5 seconds and centrifuge for 2 minutes);

(2) Install the calibration board into the instrument and run the "ROI Calibration" program;

(3) The software automatically captures images of each filter and generates ROI masks (green circles around each hole indicate successful calibration);

(4) If the image is too saturated (due to strong fluorescence inside the hole), the exposure time needs to be adjusted (such as reducing from 2048 to 1024) and the image needs to be re captured.

3. Optical calibration

Purpose: To compensate for the physical effects of optical systems (such as spectral response of filters) and ensure the accuracy of fluorescence signals;

Steps:

(1) Select the "Optical Calibration" program and load the ROI calibration file;

(2) Run the program (about 10 minutes), and the software will automatically analyze the spectral characteristics of the fluorescence signal and generate an optical correction file.

4. Dye calibration

Purpose: To verify the accuracy of fluorescence signals of different dyes (such as FAM, VIC, ROX) and ensure the reliability of multi dye experiments;

Steps:

(1) Remove the dye calibration plate (containing various fluorescent dyes such as CY3, CY5, FAM, etc.);

(2) Install the calibration board into the instrument and run the "dye calibration" program;

(3) The software automatically collects the fluorescence spectra of each dye and generates a dye calibration file (which will automatically identify dye signals in subsequent experiments).

（3） Result record and certificate

After calibration is completed, a CNAS certified calibration certificate is required, which includes the following contents:

(1) Basic information of the instrument (model, serial number, manufacturer);

(2) Calibrate environmental conditions (temperature, humidity, time);

(3) Calibration results (temperature indication error, uniformity, optical system precision, etc.);

(4) Signature of calibration personnel and auditors;

(5) Calibration institution qualification (CNAS accreditation number).

4、 Precautions for calibration

1. Calibration cycle

It is recommended to conduct a comprehensive calibration every 12 months (in accordance with JJF (Tianjin) 04-2020 requirements);

Under high usage frequency (such as running more than 10 times a day) or critical applications (such as clinical diagnosis), it can be shortened to once every 6 months;

After relocating, repairing, or replacing critical components such as heating blocks and detectors, recalibration is required.

2. Professional support

Calibration must be carried out by the manufacturer's engineer or a qualified third-party organization (such as Tianjin Institute of Metrology Supervision and Testing Science) to ensure compliance with SOP (Standard Operating Procedure);

Non professionals are prohibited from adjusting instrument parameters (such as temperature settings, optical filters) on their own to avoid affecting calibration results.

3. Verification after calibration

After calibration is completed, a standard substance verification experiment (such as plasmid DNA standard substance) needs to be run to test the repeatability (coefficient of variation ≤ 3%) and linear correlation coefficient (R ² ≥ 0.99) of Ct values to ensure calibration effectiveness;

If the verification results do not meet the requirements, recalibration or contact the manufacturer for repair is required.

5、 Common Problems and Solutions

1. The temperature uniformity does not meet the requirements

Reason: contamination of the homogenization block (such as residual PCR products), aging of the temperature sensor;

Solution: Clean the heating block (wipe with anhydrous ethanol) and replace the temperature sensor.

2. Low fluorescence signal intensity

Reason: ROI calibration failed (incorrect hole mapping), high background fluorescence;

Solution: Perform ROI calibration again and run background calibration (replace background calibration board).

3. Large variation in Ct values

Reason: Excessive temperature fluctuation (such as unstable heating block), decreased sensitivity of fluorescence detector;

Solution: Check the temperature stability of the heating block (measured with a multi-channel temperature calibrator) and replace the fluorescence detector.

VI. Summary

The calibration of fluorescence quantitative PCR instrument is a key step to ensure the accuracy and reliability of experimental data. It is necessary to strictly follow industry standards (such as JJF1527-2015, JJF (Jin) 04-2020) and regularly calibrate the temperature control system and optical system. During the calibration process, attention should be paid to environmental conditions, professional support, and result verification to ensure that the performance of the instrument meets experimental requirements. By following a standardized calibration process, experimental errors can be effectively reduced, and the accuracy and reproducibility of qPCR results can be improved.