From "failure" to precision: Does your test kit meet the sensitivity and stability standards?

In the laboratory, have you ever experienced such a scene: the nucleic acid test results fluctuate, or the performance of the test kit drops drastically after opening the bottle? The core of these problems points to the two vital points of molecular diagnostic reagents - sensitivity and stability.

As the cornerstone of precision medicine, the performance of molecular diagnostic reagents directly determines the reliability of test results. This article will reveal the evaluation principles and technical details of these two core indicators and take you into the "quality control battlefield" of scientists.

1. Definition and measurement principle of sensitivity

Sensitivity refers to the lowest concentration or minimum amount of the target that the kit can detect, and is a key indicator for measuring the detection limit.

Determination principle:

1. Dilution method: Use a known concentration of standard to perform gradient dilution until the kit cannot detect the target. The concentration at this time is the lowest limit of detection (LoD). For example, nucleic acid reagents often determine the sensitivity by diluting viral RNA or DNA samples.
2. Signal-to-noise ratio (P/N value): Some ELISA kits divide the sensitivity level by calculating the ratio of the positive sample signal to the negative control (P/N). For example, P/N ≥ 20 is high sensitivity, 10 ≤ P/N < 20 is medium sensitivity, and P/N < 10 is low sensitivity.
3. Statistical verification: Probit analysis or logistic regression was used to calculate the concentration value (LoD) corresponding to a 95% positive detection rate. Low-concentration samples need to be tested repeatedly (e.g., 20 times), and the detection rate is required to be ≥ 90% to ensure that the sensitivity meets the standard.

2. Multi-dimensional Assessment of Stability

Stability refers to the ability of a test kit to maintain its performance under different storage, transportation or use conditions, covering the following dimensions:

1. Long-term stability:
The activity retention of a reagent during its shelf life is assessed, usually by accelerated aging experiments that simulate long-term storage (such as high temperature or repeated freeze-thaw cycles).

Time point design: Store the reagents under specified conditions (such as 2~8℃ or -20℃) and test the performance regularly (such as 0, 3, 6, 12 months).

Key parameter monitoring: including sensitivity, specificity, amplification efficiency (such as PCR Ct value), reagent activity (such as enzyme activity), etc.

2. Opening stability:
The core of the uncapped stability test is to answer a question: "After the test kit is opened, how long can it last without becoming ineffective during the process of repeatedly 'breathing' air in the laboratory?"

• Repeated opening and closing simulation:

Under experimental conditions, open the reagent bottles (such as enzyme mixture, primer probe) 10 times a day for 3-7 consecutive days to simulate high-frequency usage scenarios.

• Save time limit verification:

After opening the lid, place the reagent at 4°C or room temperature, and test the performance at 0h, 8h, 24h, 48h, etc. to determine the maximum safe storage time.

Key indicators: changes in sensitivity (whether the LoD increases), whether the amplification curve is delayed/the platform value decreases.

• Environmental interference challenges:

Deliberately expose to high humidity (>80% RH) or strong light environment to detect moisture absorption or photodegradation risks. For example, fluorescent probes are easily quenched by light after opening the cover, and stability under light-proof conditions needs to be verified.

• Frequently Asked Questions:

"Invisible evaporation": The volatilization of the solution after opening the lid may cause the concentration of reagents (such as primers and probes) to increase, triggering nonspecific amplification.

“Hot or cold shock”: frequently taking out from -20℃ → opening at room temperature → refreezing can easily lead to protein denaturation or nucleic acid degradation.

3. Transportation stability:
Simulate transportation conditions such as vibration and temperature fluctuations to detect changes in reagent performance.

• Vibration test: The kit is fixed on a vibration table to simulate road transportation (frequency 5-500Hz, duration 6 hours) or air transportation turbulence (random vibration, acceleration 3Grms).

Data interpretation and improvement

“Critical value” judgment: If the sensitivity of the reagent decreases by ≤5% and the specificity remains ≥95% after transportation, it is judged to have passed.
Packaging optimization: Based on the test results, improve the shock-resistant materials (such as air column bags) or temperature-controlled packaging (phase change ice boxes).

4. Acceleration stability:
The scientific principle is the Arrhenius equation. That is, the shelf life of the reagent is "spoiled" by the extreme environment. The increase in temperature will accelerate the movement of molecules, making the degradation reaction of the reagent "fast forward". According to the Arrhenius equation, the chemical reaction rate will approximately double for every 10°C increase in temperature.

For example: If the shelf life of the reagent is 1 year at 4°C, then 1 month at 37°C ≈ 6 months at 4°C (empirical formula).

• High temperature challenge:

Condition settings: 37°C (conventional acceleration), 45°C (extreme heat shock), humidity 60%~75%.

Time gradient: test performance after 1 week, 2 weeks, and 4 weeks.

• High humidity attack: water molecules "invasion war"

Humidity control: In a humidity environment of 75%~90%, the moisture absorption of the test reagents is tested.

Fatal problems: freeze-dried powder deliquesces into clumps (such as PCR primers degrade after absorbing moisture) and enzyme activity is lost (such as proteinase K autodissolves in water).

• Light experiment: "disassembling" ultraviolet rays to test light-sensitive components (such as fluorescent probes, vitamin B12).

Test plan: Expose samples to simulated sunlight intensity (5000 lux) for 72 hours and compare them to samples kept away from light.

The actual storage life of the reagent can be deduced from the above accelerated experimental data. (Note: Some ingredients (such as proteins) may undergo irreversible denaturation at high temperatures and need to be cross-validated with long-term stability data).

5. Others

• Batch-to-batch consistency: Multiple batches of reagents need to be verified for consistency in stability.
• Differences in technology platforms: Different technologies (such as qPCR, digital PCR, and NGS) may have different sensitivity and stability assessment methods (e.g., qPCR needs to focus on primer dimers, and NGS needs to assess library stability).

3. Why are these tests so "abnormal"?

Real-life scenarios are more cruel than laboratory scenarios: express parcels may experience freezing below zero, fall from high altitudes...

The “survivor bias” trap: Only test kits that pass stress testing can avoid becoming “vase in the laboratory.”

IV. Practical Guide: How to make the test kit perform beyond its normal level?

Disassembling the "Ammunition Depot": Large package reagents should be dispensed into small tubes (e.g. 50 μL/tube) to avoid repeated freezing and thawing of the main bottle and reduce enzyme activity loss.

Temperature defense: After taking out the -20℃ reagent, quickly divide it into packages and store it at 4℃ for a short period of time (≤72h) after thawing. Repeated freezing and thawing for more than 3 times is strictly prohibited!

Marking the life and death line: Mark the date immediately after opening the lid. Discard reagents that have exceeded the storage period (such as 24 hours) directly to prevent "expired" reagents from destroying data.

Vortex + centrifuge: After thawing, vortex mix for 10 seconds and centrifuge to remove the droplets on the wall to ensure uniform concentration.

Pre-inspection to avoid rollover: Before using each batch, verify the Ct value with a positive control and immediately stop using if there is an abnormal fluctuation (such as ±2 cycles).

5. Future trends: What will a smarter test kit look like?

Multiple detection: One tube can detect 10 pathogens at the same time without compromising sensitivity!

Freeze-drying technology: no need for cold chain transportation, it can be stored at room temperature for 2 years and still remain stable10.

AI-assisted design: Algorithm optimizes primers and probes, increasing specificity by 10 times!

In conclusion:

The "sensitivity" and "stability" of reagents are the lifeline of experimental accuracy. From research and development to application, each parameter is verified and optimized countless times. Before the next experiment, you may want to take a closer look at the performance data in the manual - they may be the key to unlocking the mystery of your experimental results!