Qihengxing Blood Sample Direct PCR/qPCR Solution

In clinical testing, blood is one of the most common test samples. With the rapid development and widespread adoption of molecular testing technologies, genetic testing is now widely used for disease detection and early screening. Blood samples contain large amounts of nucleic acids, both autologous and pathogenic. Testing blood DNA/RNA can yield a wealth of information for disease screening and health management, such as genetic diseases, early disease screening (e.g., cancer screening), infectious disease detection, and forensic identification.

In the field of molecular diagnostics, commonly used detection methods include PCR, qPCR, and next-generation sequencing. Currently, conventional detection methods involve extracting and purifying nucleic acids, followed by downstream testing. For example, DNA extraction takes 1-2 hours, and PCR/qPCR testing takes approximately 1 hour, for a total of approximately 3 hours.

To improve the efficiency of blood testing, a method has emerged that directly performs PCR/qPCR on blood after pretreatment. However, this method is limited in its application due to factors such as excessive sample pretreatment, low amplification efficiency, and poor resistance to inhibition.

Beijing Qihengxing Biotechnology has achieved efficient PCR/qPCR testing of various blood samples after simple lysis by constructing and screening genetically engineered enzymes and optimizing amplification buffers. The entire experimental process is shortened to 1 hour, while also having excellent anti-interference capabilities and stable amplification efficiency.

Direct PCR in blood Premix ( StarLighter Blood Direct PCR Mix )

StarLighter Blood Direct PCR Mix utilizes a unique PCR buffer system that effectively reduces the effects of inhibitors in the blood on amplification. It allows direct PCR using whole blood samples as templates without the need for DNA purification or sample processing. It is suitable for EDTA-anticoagulated blood samples stored under different conditions and can be used for single or multiplex direct blood amplification.

Product advantages and experimental data

1. Suitable for multiple amplification, compatible with templates of different GC% contents, good amplification uniformity, and a wide range of blood input amounts.

Fig2.1 Direct amplification of 5%, 10%, 15%, 20%, 25%, and 30% fresh EDTA anticoagulated blood using hexaplex primers was performed using the blood direct amplification kits from Qihengxing and supplier T. The amplified fragment ranged from 200 to 800 bp, and the GC% ranged from 35% to 77%. Amplification was performed for 35 cycles, and the PCR products were detected by agarose gel electrophoresis.
The results showed that the Qihengxing blood direct amplification kit was superior to supplier T in terms of product quantity and amplification uniformity.

Fig 2.2 Direct amplification results of EDTA anticoagulated blood stored at room temperature

For EDTA blood samples stored at different conditions such as room temperature, 4°C, and -20°C, the StarLighter Blood Direct PCR Mix can accurately amplify the products of six items.

Fig 2.3 Direct amplification results of EDTA anticoagulated blood stored at 4°C and -20°C

PART TWO Simple blood lysis direct expansion qPCR reagents box (StarLighter Probe qPCR Kit for Blood)

StarLighter Probe qPCR Kit for Blood It is suitable for various types of blood samples and does not require tedious nucleic acid extraction and purification. qPCR amplification can be performed by simply lysing the blood sample and taking the supernatant. The operation is simple and can reduce the risk of contamination. The test results are consistent with those obtained by using the pure DNA product after nucleic acid extraction as a template.

Product advantages and experimental data

1. Fast operation, from blood sample to complete genotyping/quantification analysis , Solve big problems in 1 hour .

Fig 3.1 StarLighter Probe qPCR Kit for Blood operation flow chart

2. SNP typing is accurate, Compatible with various types of samples : Fresh blood sample A and refrigerated (stored at 4°C for 2 months) blood sample A genotyped with known SNPs Three gene loci Test, results Both Sanger sequencing is completely consistent .

Fig. 3.2 Detection results for different SNPs in fresh blood sample A after lysis and release. Blood was lysed and centrifuged, and 4 μL of the supernatant was added to a 20 μL reaction system as a template for qPCR. The results were consistent with those from Sanger sequencing.

Fig. 3.3 Detection results for different SNPs after lysis and release of stored blood sample A. Blood was lysed and centrifuged, and 4 μL of the supernatant was added to a 20 μL reaction system as a template for qPCR. The results were consistent with those from Sanger sequencing.

3. Test results and carry After purification, qPCR was performed Consistency : For the same sample, StarLighter Probe qPCR Kit for Blood Reagent test kit and tradition Blood liquid Genomic DNA extraction kit Then qPCR and other two methods were used to Specific gene loci The test results show two Methods of detection Ct value Very consistent with the fluorescence intensity .

Fig 3.4 Comparison of qPCR data after extraction using the Qihengxing StarLighter Probe qPCR Kit for Blood and a traditional blood nucleic acid extraction kit. The thick line represents the qPCR results after treatment with nucleic acid release solution, while the thin line represents the qPCR results after whole blood genomic DNA extraction and purification. Blue represents the FAM channel, orange represents the Rox channel (internal control), and green represents the Vic channel.

From technological breakthroughs to application scenarios, Qihengxing Bio has integrated multi-platform product development experience such as sample pre-treatment, core enzyme development, and buffer system optimization, and has created a full-process solution centered on direct amplification of blood DNA after simple lysis. Whether it is the high-purity guarantee of automated/manual whole blood extraction, the minimalist breakthrough of direct amplification PCR, or the high efficiency and accuracy of rapid lysis qPCR, each technology embodies a deep insight into the industry's pain points and innovative practices. In the future, Qihengxing will continue to work hard to promote the expansion of blood gene testing technology to deeper and broader dimensions, promote the optimization of the entire process from sample processing to precise analysis, and provide product application support for more application scenarios.