When nucleic acid extraction becomes history, the "minimalist revolution" of molecular diagnostics is coming
No need for centrifuges or pipettes—blood direct amplification PCR/qPCR technology is redefining the molecular diagnostic process with its disruptive logic of “original sample in, amplified product out.” This technology not only reduces the number of experimental steps by 60%, but also [1] , and its "hardcore" strength in combating the complex matrix of blood has taken the field by storm in areas such as early cancer screening and pathogen detection. This article will delve into why direct blood amplification technology has been called "the victory of inhibitory polymerases" and what "offensive and defensive battles" it faces in clinical translation.
1. Technological breakthrough: a paradigm shift from “sample pretreatment” to “inhibitor tolerance”
The pain point of traditional PCR is the "sample purification dependency": PCR inhibitors such as hemoglobin and lactoferrin must be removed by centrifugation, magnetic bead adsorption, etc. (for example, hemoglobin > 2 mg/mL can inhibit Taq enzyme activity). [2] ). Direct blood expansion technology achieves a "hands-free breakthrough" through a three-pronged strategy:
Inhibition-resistant polymerases: Mutants derived from directed evolution (e.g., Thermo Fisher's Platinum™ Direct PCR Universal Mix) can tolerate 8% whole blood addition and maintain >90% activity even at heparin concentrations of 1 U/μL. [3] ;
Cofactor optimization: Add stabilizers such as trehalose and BSA to reduce the probability of inhibitor binding to the enzyme active center.
One-step lysis buffer: contains a chaotropic salt (such as guanidine thiocyanate) and a non-ionic surfactant (Triton X-100) to simultaneously achieve red blood cell lysis and protein denaturation;
pH control technology: The reaction system is maintained at pH 8.5-9.0 to weaken the quenching effect of the iron porphyrin structure of hemoglobin on fluorescence.
Hairpin structure blocking: introducing a phosphorothioate bond modification at the 3' end of the primer to reduce nonspecific amplification caused by single-stranded DNA binding proteins in the blood;
GC clamp strategy: Design a 5'-end GC clamp for targets with high GC content to improve primer annealing efficiency in complex matrices.
Direct amplification qPCR in blood enables real-time quantitative detection of target nucleic acids, monitoring the fluorescence signal in real time during the amplification process and accurately quantifying the concentration of the target nucleic acid. Currently, several mature direct amplification qPCR kits in blood are available on the market, with detection sensitivities reaching 10 copies/μL and specificities exceeding 99%. These include companies such as Maidian, Zhuhai Baorui, and Beijing Qihengxing Biotechnology.
2. Clinical Evidence: When Sensitivity Exceeds 1% VAF, Liquid Biopsy Enters a New Inflection Point
Blood direct amplification PCR/qPCR technology has, in a sense, promoted the progress of molecular diagnostic POCT. In clinical validation, blood direct amplification technology has demonstrated "disruptive compatibility":
Typical cases:
★Novel coronavirus load monitoring: Using the Brilliance Direct SARS-CoV-2 RT-qPCR Kit, the test of 50 clinical samples showed that the correlation with the Ct value of the nucleic acid extraction method was R²=0.98 [5] ;
Post-transplant CMV virus early warning: A team from Peking University People's Hospital used direct blood amplification technology to advance the CMV DNA detection window by 3 days, preemptively initiating antiviral treatment and reducing the incidence of rejection by 27%. [6] .
3. Technical limitations: advantages and disadvantages depend on each other, so we should treat them dialectically
Direct amplification qPCR (qPCR) in blood can achieve a sensitivity of 10 copies/μL, but it can still miss detections at extremely low concentrations of target nucleic acids. Regarding specificity, issues such as nonspecific amplification and primer dimers have not yet been fully resolved, especially when multiplexing, which increases the risk of cross-reactions. Furthermore, individual differences in blood composition pose a challenge to direct amplification reagent systems.
2. The "Rashomon" of Standardization: Standardization and quality control are the main operational challenges facing direct blood amplification technology. Kits from different manufacturers vary significantly in performance, and there is a lack of unified quality control standards. Even minor differences in the operational process, such as sample volume and mixing level, can affect the reproducibility of test results.
3. Extreme samples and the dilemma of “crosstalk between multiple detection signals”
In terms of clinical applicability, the direct amplification technology of blood has poor detection effects on certain special samples. For example, for samples with severe hemolysis, hyperlipidemia or jaundice, the detection accuracy may drop significantly. In tumor ctDNA detection, the ctDNA concentration of early tumor patients is extremely low, making detection more difficult. At the same time, the complexity of result interpretation cannot be ignored. Fluorescence curve analysis of direct amplification qPCR of blood requires professional knowledge and experience, especially in the judgment of weak positive results, which is subjective. In multiple detections, signal interference between different targets may increase the risk of misjudgment of results.
IV. Conclusion The "decentralized" revolution in molecular diagnostics is underway
While direct amplification PCR and qPCR technologies in blood have achieved significant breakthroughs in molecular diagnostics, they still face numerous challenges. These limitations will need to be overcome through technological innovation and method optimization, such as developing more efficient inhibitor-tolerant enzyme systems, establishing standardized operating procedures, and introducing artificial intelligence to assist in result analysis. Direct amplification technology in blood not only simplifies experimental procedures but also heralds the migration of molecular diagnostics from centralized laboratories to bedside, community, and even home settings. When the cost of testing exceeds the critical point of 100 yuan per test, we may witness the transformation of early cancer screening from a "high-end physical examination" to a "routine item," and the shift of pathogen detection from "passive medical treatment" to "active monitoring."
[1] Data source: Clinical Chemistry 68(4): 551-560 (2022)
[2] See Journal of Molecular Diagnostics 19(2): 234-241
[3] Quoted from Thermo Fisher Scientific Technical White Paper TB-0017-EN
[4] Based on NCCLS EP17-A2 standard verification data
[5] Brilliance Biopharmaceuticals Clinical Trial Report HCY-2023-0012
[6] Unpublished data from the Peking University People’s Hospital research team
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