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A In the process of system configuration, components such as primers and enzymes are in excess, but templates are not in excess. Template DNA molecules randomly enter each small reaction system. After PCR amplification, the system containing the template completes the amplification, and the system does not contain the template without amplification.
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A Cause: The strain is mutated, and the current probe is not suitable for detection; the nucleic acid caused by the extraction method (failure to completely extract the bacteria, fungi, etc. included in the sample) cannot be detected;
Exclusion verification: DNA extraction from culture, followed by amplification with our probes, can be verified; it is recommended that blood culture and nucleic acid testing be the same blood sample.
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A Judgment standard ≥ 3 positive droplets, but for the positive droplets at the critical point, it is necessary to carry out in- depth analysis in combination with negative/ positive quality control materials . When there is a vertical line of signals , it is judged as a non-positive signal.
low-concentration samples, a higher multiple buffer can be selected for sample loading adjustment .
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A Plasma : ≥ 2ml is recommended, the components are relatively simple, and the contamination of human DNA is removed. The nucleic acid composition is simpler, and the bacterial nucleic acid concentration is higher, which is convenient for detection.
Note : The rotating speed should not be too high during centrifugation, otherwise it will easily lead to the loss of intracellular bacteria and affect the final test result;
Whole blood: ≥4ml is recommended, the components are relatively complete, and the consistency with blood culture specimens is good, but there is contamination of human DNA.
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A Bacteria (Gram-positive bacteria, Gram-negative bacteria), invasive fungi, viruses. At the same time, drug resistance gene detection can also be carried out.
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A It is recommended to set up a control experiment for division. When not set, generally use the fluorescence intensity of 3000 as the threshold point, or use the negative cluster as a reference to judge the positive. You can refer to the experience of flow cytometry to make a division.
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A Fluorescence quantification is based on the amplification curve. The detection signal of each cycle will be calculated by the logarithm of 2 to the N power, which can only be used to distinguish the difference of more than 2 times. The digital PCR system can detect gene expression differences as low as 0.1-fold by the Poisson distribution calculation formula .
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A For mutation frequency, digital PCR can reach 0.01% or less , and single copy can be achieved for microorganisms; RT-PCR can only achieve 1%.
