How to inactivate 2019-nCoV specimens? Will it affect the test result?
It is recommended to inactivate specimens by water bath or dry bath (oven) at 56℃ for 30-45 minutes. In case of dry bath, the inactivation time for heating specimens to56℃can be extended appropriately (by testing the time needed for heating the same volume of normal saline to 56℃).According to our observation, inactivation has a little impact on the subsequent test result.
How to arrange three negative controls to check the contamination in the experiment?
According to the requirement of test, three negative controls should be set to check the contamination. We can make one negative control, one blank control with water and an environmental sample control in the whole batch of specimens so as to meet the requirements of three controls.
How to deal with negative and positive controls of the 2019-nCoV RNA detection kit from Da An Gene?
The instruction for use of the kit recommends participation in extraction of negative and positive controls. Negative control must participate in the extraction for monitoring the pollution of laboratory environment. Normal saline can be used as the control to monitor the pollution of the whole experiment process.
What is the name of human housekeeping gene in this kit?
Its name is RNase P (ribonuclease P). RNase P is ubiquitous in all organs, tissues and cells of the human body, and it can be detected from RNA fragments of human specimens. Meanwhile, it can also be used as an internal standard, and monitor the accuracy of the whole experimental operation process.
How to extract viral nucleic acid withthe2019-nCoV RNA detection kit from Da An Gene? What methods can be used?
There are column extraction method, paramagnetic particle method and one-step extraction method for RNA virus extraction. The paramagnetic particle method and column extraction method can be divided into manual operation and automatic machine operation. Generally, original liquid specimens are taken for extraction. The automatic paramagnetic particle extraction is characterized to be the best effect, with the advantages such as safe and convenient operation, large extraction amount for one time, high extraction efficiency and the best subsequent amplification effect. Therefore, it is recommended to use the paramagnetic particle method with automatic machine to extract 2019-nCoV nucleic acid for the subsequent PCR amplification experiment. The specific instrument and magnetic particle reagent are with reference to the instructions of each manufacturer.
In addition to ABI7500, Viia7,QS DX instruments, can Bio-Rad CFX96, Hongshi SLAN96, AGS4800, Agilent Mx3000p and Mx3005P, ABI7300plus and Q3 be used for the 2019-nCoV RNA detection kit from Da An Gene?
Bio-Rad CFX96, Hongshi SLAN96, AGS4800, Agilent Mx3000p and Mx3005P can be used for the 2019-nCoV RNA detection kit from Da An. ABI7300plus and Q3 cannot be used for 2019-nCoV reagents of Da An Gene due to lack of CY5 channel. Theoretically, instruments with the three channels (FAM, VIC(HEX) and CY5) can be used for 2019-nCoV reagents of Da An Gene, and those fail to meet the requirements cannot be used for 2019-nCoV reagents of Da An Gene.
Why do the instructions for the 2019-nCoV RNA detection kit from Da An Gene mention the target genes corresponding to FAM, VIC and CY5 channels?
In the "test principle" of the instructions, target genes corresponding to three channels are introduced. N gene probe is labeled with FAM, ORF1ab gene is labeled with Yellow fluorescence, and can be tested with VIC passage, so the description of channel in result interpretation is VIC channel. The internal standard gene probe is labeled with Cy5.
How to interpret the test results when it is inconsistent with the clinical diagnosis of 2019-nCoV?
As RNA virus, 2019-nCoV is easily degraded, and the test results are also related to sampling, nucleic acid extraction and the nucleic acid concentration of the specimens, so all of these factors need to be analyzed one by one. Meanwhile, the results can be interpreted in combination with clinical symptoms.
How to deal with the problem when there is no internal standard tested in specimen by using 2019-nCoV reagents of Da An Gene?
If there is no internal standard tested in specimen by using 2019-nCoV reagents of Da An Gene, the specimen must be reviewed. It can be remixed to extract nucleic acid. If there is still no internal standard tested by 2019-nCoV reagents of Da An Gene ,the specimen is unqualified when there is no extraction problem, and the clinical re-sampling and review shall be notified.
How to confirm positive cases by laboratory ?
The real-time fluorescent RT-PCR test results of 2 targets (ORF1ab and N) of 2019-nCoV in the same specimens are tested as positive. If there is a positive test result for a single target, it needs to be re-sampled and re-tested.
What are the reasons of false negatives and false positives in 2019-nCoV nucleic acid test? How to avoid it?
Reasons for false negative: No suitable specimens were collected in different courses of disease; virus from collected specimens were not enough to be detected; it is not sensitive enough for the test reagent; the nucleic acid extraction is not efficient. False positive: There are cross contamination during nucleic acid extraction and PCR products contamination; it is also related to specimen type and sampling method. Broncho-alveolar lavage fluid is better than swab, and can be collected simultaneously with nasopharyngeal swabs.