Quality control kit of nucleic acid detection system and quality control method and application of quality control kit

Abstract

The invention provides a quality control kit of a nucleic acid detection system as well as a quality control method and application of the quality control kit. The quality control kit comprises the coliphage MS2 and a first sequence group. The first sequence group comprises a forward primer of a sequence as shown in SEQ IQ NO: 1, a reverse primer of a sequence as shown in SEQ IQ NO: 2 and a probe of a sequence as shown in SEQ IQ NO: 3. The quality control method comprises the following steps: mixing a specimen with bacteriophage MS2, and carrying out nucleic acid extraction to obtain an eluent; and mixing an eluent with the feline blood plasma bacterial plastid, the first sequence group and the second sequence group, and carrying out multiple real-time polymerase chain reaction. The quality control kit of the nucleic acid detection system is used for evaluating the recovery rate of nucleic acid and the whole-process quality control of the nucleic acid detection system.

Description

Technical Field

[0001] The present invention relates to a nucleic acid detection quality control kit, particularly to a nucleic acid detection quality control kit comprising primer pairs and probes for Escherichia coli phage MS2 and primer pairs and probes for Mycoplasma haemofelis Mh; the present invention also relates to a detection method for the nucleic acid detection quality control kit; and the present invention also relates to a use of the quality control kit. Background Art

[0002] The prior art uses point of care - nucleic acid testing (POC - NAT) devices to detect viral nucleic acids in the human body or for research and diagnosis of diseases by real - time polymerase chain reaction (real - time PCR). Monitoring whether the entire process steps of POC - NAT are normal includes (1) monitoring the efficiency of nucleic acid extraction, such as monitoring the recovery rate of nucleic acid extraction and the residual interference substances after nucleic acid extraction, and (2) monitoring the qPCR process, such as the heating of qPCR and the reception of fluorescence signals, and the enzyme performance. The efficiency of nucleic acid extraction will affect the sensitivity and stability of POC - NAT, and even lead to false negative results.

[0003] Based on the convenience of storing positive standards, the prior art adds exogenous DNA templates to the lysis buffer before nucleic acid extraction to serve as positive controls for the nucleic acid extraction process. In addition, the successful amplification result of subsequent PCR can be compared with the successful extraction of nucleic acids and prove that there is no high - concentration residual interference substance during nucleic acid extraction. Alternatively, the exogenous DNA template can also be directly incorporated into the PCR reaction to confirm whether the test sample can support qPCR amplification. However, the method of directly adding exogenous DNA templates to the PCR reaction will cause the breakage and degradation of DNA templates under the conditions of the lysis solution environment and the presence of physical factors (heating and oscillation) due to the lack of envelope protection, thereby leading to a decrease in the recovery rate. Therefore, this method for monitoring POC - NAT cannot effectively verify the recovery rate of nucleic acid extraction.

[0004] In addition, since most of the prior art performs the quality control of POC - NAT through a single qPCR, it is impossible to distinguish in a single reaction whether the reason for the Cq delay is due to poor recovery rate of nucleic acid extraction or excessive residual interference substances.

[0005] Therefore, it is an urgent problem in the art to develop a nucleic acid detection quality control kit that can distinguish whether the abnormal signal of the nucleic acid detection system comes from problems at the nucleic acid extraction end (for example, abnormal recovery rate of nucleic acid extraction or residual interference substances after nucleic acid extraction) or from problems at the qPCR end (for example, heating of qPCR and reception of fluorescence signals, and abnormal enzyme performance) to improve the sensitivity and stability of the nucleic acid detection system. Summary of the Invention

[0006] To solve the problems of the above-mentioned prior art, the object of the present invention is to provide a quality control kit for a nucleic acid detection system, by using Enterobacteria phage MS2 RNA and its sequence group, as well as Mycoplasma haemofelis plastid and its sequence group, so as to achieve the purpose of quality control of the nucleic acid detection system.

[0007] Another object of the present invention is to provide a quality control method for a nucleic acid detection system, by using multiplex real-time polymerase chain reaction, to achieve the purpose of distinguishing whether the abnormal signal of the nucleic acid detection system comes from problems at the nucleic acid extraction end (for example, abnormal nucleic acid extraction recovery rate, or residue of interfering substances after nucleic acid extraction) or from problems at the qPCR end (for example, heating of qPCR and reception of fluorescence signals).

[0008] Another object of the present invention is to provide a use of a quality control kit for a nucleic acid detection system.

[0009] To achieve the above object, the present invention provides a quality control kit for a nucleic acid detection system, including Enterobacteria phage MS2 and a first sequence group. The first sequence group includes a forward primer having a sequence as shown in SEQ IQ NO:1; a reverse primer having a sequence as shown in SEQ IQ NO:2; and a probe having a sequence as shown in SEQ IQ NO:3.

[0010] In a specific embodiment, the quality control kit for the nucleic acid detection system further includes Mycoplasma haemofelis Mh plastid and a second sequence group. The second sequence group includes a forward primer having a sequence as shown in SEQ IQ NO:4; a reverse primer having a sequence as shown in SEQ IQ NO:5; and a probe having a sequence as shown in SEQ IQ NO:6.

[0011] The present invention also provides a quality control method for a nucleic acid detection system, including the steps of: Step S10: Provide a quality control kit for the nucleic acid detection system; Step S20: Provide a sample; Step S30: Mix the sample with Enterobacteria phage MS2 particles with a known concentration in an extraction cartridge of the nucleic acid detection system, and perform nucleic acid extraction to obtain an eluate; Step S40: Mix the eluate, Mycoplasma haemofelis Mh plastid, the first sequence group and the second sequence group, and perform a first multiplex real-time polymerase chain reaction to obtain a first MS2 Cq value and a first Mh Cq value; and Step S50: Determine the recovery efficiency of nucleic acid extraction based on the first MS2 Cq value.

[0012] In a specific embodiment, this quality control system is paired with a Bio-Rad CFX96 real-time quantitative polymerase chain reaction instrument. First, a standard curve of Escherichia coli phage MS2 RNA is established, and its quantitative linear range is between 10^5 and 10^1 copies.

[0013] In a specific embodiment, the amount of Mycoplasma haemofelis Mh plasmid in each reaction is 10^3 copies.

[0014] In a specific embodiment, the quality control method of the nucleic acid detection system further includes the steps of: Step S60: Put the quantified Escherichia coli phage MS2 into extraction, mix the Mycoplasma haemofelis Mh plasmid, the first sequence group and the second sequence group, and perform multiplex real-time polymerase chain reaction to obtain the second Mh Cq value; when the difference between the second Mh Cq value and the first Mh Cq value is less than or equal to 1, there is no interference inhibitor in the eluate; when the difference between the second Mh Cq value and the first Mh Cq value is greater than 1, there is an interference inhibitor in the eluate.

[0015] In a specific embodiment, the quality control method of the nucleic acid detection system further includes the following steps: Step S71: Dilute the eluate 10-fold to obtain a 10-fold diluted eluate; and Step S72: Mix the undiluted and 10-fold diluted eluates, the Mycoplasma haemofelis Mh plasmid, the first sequence group and the second sequence group respectively, and perform multiplex real-time polymerase chain reaction to obtain the second MS2 Cq value; when the difference between the MS2 Cq value of the undiluted eluate (i.e., the first MS2 Cq value) and the MS2 Cq value of the 10-fold diluted eluate (i.e., the second MS2 Cq value) is greater than or equal to 3, when the difference between the MS2 Cq value of the undiluted eluate (i.e., the first MS2 Cq value) and the MS2 Cq value of the 10-fold diluted eluate (i.e., the second MS2 Cq value) is less than 3, there is an interference inhibitor in the eluate.

[0016] In a specific embodiment, the interference inhibitor includes inhibitors in the eluate.

[0017] In a specific embodiment, the upper limit of the DNA concentration of the sample is 200 ng.

[0018] In a specific embodiment, the quality control method of the nucleic acid detection system further includes the following steps: Step S81: Repeat the steps of Step S10 to Step S40 multiple times to obtain multiple MS2 Cq values and multiple Mh Cq values, and calculate the average value of the multiple MS2 Cq values and the standard deviation of the multiple MS2 Cq values, as well as the average value of the multiple Mh Cq values and the standard deviation of the multiple Mh Cq values; wherein the standard range of the quality control of the nucleic acid detection system includes the average value of the MS2 Cq values + / - 2 x the standard deviation of the MS2 Cq values, and the average value of the Mh Cq values + / - 2 x the standard deviation of the Mh Cq values; and Step S82: When the obtained MS2 Cq value or Mh Cq value is not within the standard range, it is determined that the detection fails to meet the quality control standard.

[0019] In a specific embodiment, the nucleic acid detection system includes a fully automatic nucleic acid detection system, such as a commercial brand: Dagene G1 fully automatic nucleic acid detection system.

[0020] The present invention provides a use of a quality control kit for a nucleic acid detection system, and the quality control kit for the nucleic acid detection system is used to evaluate the recovery rate of a sample after nucleic acid extraction, whether there is residual interference inhibitor in the eluate obtained after nucleic acid extraction of the sample, the quality control of the extraction cartridge of the nucleic acid detection system, or the quality control of the instrument of the nucleic acid detection system.

[0021] The advantages of the present invention are as follows:

[0022] High specificity: The MS2 forward primer shown in SEQ ID NO:1, the MS2 reverse primer shown in SEQ ID NO:2, and the MS2 probe shown in SEQ ID NO:3 from Escherichia coli phage MS2, and the Mh forward primer shown in SEQ ID NO:4, the Mh reverse primer shown in SEQ ID NO:5, and the Mh probe shown in SEQ ID NO:6 from Mycoplasma haemofelis of the quality control kit of the present invention have high specificity and cannot identify the sequences of other species. MS2 phage mainly exists in polluted wastewater. The designed primers and probes are developed and designed based on biological information analysis by comparing a large range of NCBI Database sequences. The Primer-Blast results will not identify other species. The paired Mycoplasma haemofelis primers and probes, whose pathogen is mainly pets, also have good specificity in the Primer-Blast analysis results and will not identify other sequences. These two species do not exist in the experimental environment or reagent formula, nor do they belong to human pathogen nucleic acid detection products. Therefore, they can be used for the factory quality control process. When actually applied to the quality control of the G1 fully automatic nucleic acid detection system, no non-specific signals will be generated.

[0023] Multi - functions and Applications: The MS2 forward primer shown in SEQ ID NO:1, the MS2 reverse primer shown in SEQ ID NO:2, and the MS2 probe shown in SEQ ID NO:3 can be used as standards for nucleic acid extraction in the G1 extraction cartridge of the Dagene G1 fully automatic nucleic acid detection system to evaluate the recovery rate of nucleic acid extraction; and the Mh forward primer shown in SEQ ID NO:4, the Mh reverse primer shown in SEQ ID NO:5, and the Mh probe shown in SEQ ID NO:6 can be used to evaluate whether there are interference and inhibition factors in the G1 extraction cartridge and as an endogenous control for qPCR reagents. Therefore, this quality control kit can detect the nucleic acid extraction recovery rate of the G1 extraction cartridge of the Dagene G1 fully automatic nucleic acid detection system, evaluate whether there are interference and inhibition factors in the eluate obtained after nucleic acid extraction of the sample, the quality control of the extraction cartridge of this nucleic acid detection system, or the quality control of the instrument of this nucleic acid detection system through multiplex qPCR reactions.

[0024] Not easily interfered by the nucleic acid concentration of the human genome: The upper tolerance limit of human genomic DNA in this multiplex qPCR reaction is 200 ng. Therefore, when the quality control kit is used to detect samples from the human upper respiratory tract, the qPCR reaction is not easily affected by the sample concentration.

[0025] High sensitivity: The detection rate of 10 copy MS2 RNA / rxn is 100% in both the G1 fully automatic nucleic acid detection system and the Bio Rad system. For extraction systems with poor recovery rates, the recovery rate can be estimated. Description of the Drawings

[0026] Figure 1 It is a flowchart of the quality control method for the nucleic acid detection system of the present invention. Figure 2 It is a result graph of the linear range of the Escherichia coli phage MS2 RNA quantitative standard curve of the multiplex real - time polymerase chain reaction of the quality control system of the present invention. Figure 3 It is a result graph of the amplification curves of MS2 RNA serial dilution and quantitative Mh plasmid nucleic acid amplification of the multiplex real - time polymerase chain reaction of the quality control system of the present invention, and it is the same experiment as above Figure 2 for the same experiment. Figure 4 It shows the result graph of the nucleic acid amplification curves of two amplification products of the multiplex real - time polymerase chain reaction of the present invention at the same concentration. Figure 5 It shows the result graph of the sensitivity test of the multiplex real - time polymerase chain reaction of the quality control of the present invention. Figure 6 It is a flowchart of the quality control method for the nucleic acid detection system of the present invention. Figure 7 It is a flowchart of the quality control method for the nucleic acid detection system of the present invention. Figure 8 It is a flowchart of the quality control method for the nucleic acid detection system of the present invention. Detailed implementation manners

[0027] The following describes the implementation manners of the present invention through specific specific examples. Those of ordinary skill in the art can understand other advantages and effects of the present invention through the content shown in this specification. However, the exemplary embodiments shown in the present invention are only for illustrative purposes and should not be regarded as limiting the scope of the present invention. In other words, the present invention can also be implemented or applied through other different specific examples, and various details in this specification can also be modified and changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0028] Unless otherwise specified herein, the singular forms "a" and "the" used in the specification and the appended patent claims include plural entities.

[0029] Unless otherwise specified herein, the term "or" used in the specification and the appended patent claims includes the meaning of "and / or".

[0030] Preparation Example 1: Design of primer pairs and probes for Escherichia coli phage MS2 (Enterobacteriaphage MS2) for quality control of nucleic acid detection systems

[0031] In the region of the coat protein across the lysis protein (between 1661 bp and 1760 bp of the genome of Escherichia coli phage MS2 (hereinafter referred to as "MS2"), the gene accession number in the NCBI gene database: EF204940.1), an MS2 forward primer shown in SEQ ID NO:1 (between 1661 bp and 1683 bp of the genome of MS2), an MS2 reverse primer shown in SEQ ID NO:2 (between 1738 bp and 1760 bp of the genome of MS2), and an MS2 probe shown in SEQ ID NO:3 (between 1692 bp and 1716 bp of the genome of MS2) were designed. The fragment size after polymerase chain reaction (PCR) amplification was 100 bp. The NCBI Primer-BLAST was used to test the specificity of the forward primer shown in SEQ ID NO:1, the reverse primer shown in SEQ ID NO:2, and the probe shown in SEQ ID NO:3. The results confirmed that the forward primer shown in SEQ ID NO:1, the reverse primer shown in SEQ ID NO:2, and the probe shown in SEQ ID NO:3 had species specificity and could not perform non-specific amplification with other species.

[0032] Preparation Example 2 Primers and Probes for Mycoplasma haemofelis Designed for Quality Control of Nucleic Acid Detection Systems

[0033] In the 16S rRNA of Mycoplasma haemofelis (hereinafter referred to as "Mh") (Gene Accession Number in NCBI Gene Database: JQ689951.1), an Mh forward primer as shown in SEQ ID NO:4 (located between 563bp and 583bp of the 16S rRNA gene of Mh), an Mh reverse primer as shown in SEQ ID NO:5 (located between 691bp and 710bp of the 16S rRNA gene of Mh), and an Mh probe as shown in SEQ ID NO:6 (located between 663bp and 686bp of the 16S rRNA gene of Mh) were designed. The fragment size after PCR amplification is 148bp. The specificity of the forward primer as shown in SEQ ID NO:4, the reverse primer as shown in SEQ ID NO:5, and the probe as shown in SEQ ID NO:6 was verified using NCBI Primer-BLAST. The results confirmed that the forward primer as shown in SEQ ID NO:4, the reverse primer as shown in SEQ ID NO:5, and the probe as shown in SEQ ID NO:6 have species specificity and cannot perform non-specific amplification with other species.

[0034] See Figure 1 , one aspect of the present invention provides a quality control method for a nucleic acid detection system, including the steps: Step S10: Provide a quality control kit for the nucleic acid detection system, including a multiplex real-time polymerase chain reaction reagent containing Escherichia coli phage MS2 with a known concentration, a mixture of the first MS2 sequence combination and the second Mycoplasma haemofelis sequence group, and a Mycoplasma haemofelis plasmid with a fixed concentration; Step S20: Provide a sample; Step S30: Mix the sample with Escherichia coli phage MS2 with a known concentration and perform nucleic acid extraction to obtain an eluate. Step S40: The eluate is further mixed with the multiplex real-time polymerase chain reaction reagent containing the mixture of the first MS2 sequence combination and the second Mycoplasma haemofelis sequence group and the Mycoplasma haemofelis plasmid with a fixed concentration to obtain a first MS2 Cq value and a first Mh Cq value. Step S50: Determine the recovery efficiency of nucleic acid extraction based on the first MS2 Cq value.

[0035] Example 1 Sensitivity test of MS2 / Mh multiplex real-time polymerase chain reaction (hereinafter referred to as "multiplex qPCR") reagent

[0036] Mix 1x TOYOBO master mix, 1 μM MS2 forward primer, 1 μM MS2 reverse primer, 0.25 μM MS2 probe, 0.25 μM Mh forward primer, 0.25 μM Mh reverse primer, 0.2 μM Mh probe and 10^3 copies (cp) of Mh DNA plasmid to obtain a multiplex qPCR reaction mixture with a volume of 20 μL.

[0037] Multiplex qPCR is performed on a BioRad real-time PCR instrument (CFX-96) or a POCT rapid nucleic acid detection instrument, the Dagene G1 fully automated nucleic acid detection system, and the reaction conditions are: 60 °C for 5 minutes; 95 °C for 30 seconds; repeat 45 cycles of 95 °C for 5 seconds and 60 °C for 5 seconds.

[0038] When multiplex qPCR is tested on a BioRad real-time PCR instrument, a quantitative standard curve needs to be prepared. In each multiplex qPCR reaction, the amount of Mh DNA plasmid in the multiplex qPCR mixture is fixed at 10^3 copies as the quality control concentration of the multiplex qPCR reagent, and its Cq can be used as the interference interpretation standard. In addition, MS2 RNA is serially diluted 10-fold, and 10, 10^2, 10^3, 10^4, 10^5 copies of MS2 RNA are added to the multiplex qPCR mixture for multiplex qPCR reaction, and its Cq can be used to evaluate the nucleic acid recovery efficiency. When performed on the Dagene G1 fully automated nucleic acid detection system, the Cq of MS2 or Mh at a fixed concentration is stable.

[0039] The results of the Cq values of the obtained MS2 / Mh multiplex qPCR reactions are shown in Table 1. The Cq value obtained from 10^5 copies of MS2 RNA is the lowest, and the Cq value obtained from 10 copies of MS2 RNA is the highest. Moreover, the Cq values obtained from MS2 RNA with a 10-fold difference are approximately 3 different, which is in line with expectations. In addition, the amount of Mh DNA plasmid in each reaction is fixed at 10^3 copies, so the obtained Cq values are all approximately 28.5, which is in line with expectations, thus demonstrating the accuracy of the present invention.

[0040] See Figure 2 , the results of the quantitative standard curve show that R2 is 0.995, the slope is -3.591, and the efficiency is 90%. Therefore, the quantitative credible range is 10^5 to 10^1 copies of MS2 RNA. In addition, see Figure 3 , the amplification curve shows that the multiplex qPCR reaction without added MS2 RNA (no template control, NTC) did not amplify any products.

[0041] Table 1: Cq values of MS2 / Mh multiplex qPCR reactions

[0042] To confirm whether stable results are presented for 10^1 copies of MS2 RNA in the MS2 / Mh multiplex qPCR reaction, the Mh plastid was maintained at 10^3 copies, and the reaction was repeated 8 times for 10^1 copies of MS2 RNA and 2 times for 10^3 copies of MS2 RNA. See Table 2, Figure 4 and Figure 5 , the results showed that the Cq values of 10^3 copies of MS2 RNA were extremely stable between 30.1 - 30.3. Even with only 10^1 copies of MS2 RNA, it could still be successfully detected by the primers of the present invention. That is, the sensitivity of MS2 RNA in the MS2 / Mh multiplex qPCR reaction of the present invention was ≤10 copies. As for the amplification curve, it showed that no products were amplified in the multiplex qPCR reaction (NTC) without adding MS2 RNA. In addition, the Mh plastid maintained at 10^3 copies did not affect the sensitivity of MS2 RNA. Furthermore, there was no competitive relationship between MS2 RNA and the Mh plastid in the MS2 / Mh multiplex qPCR reaction, and their signals did not interfere with each other.

[0043] Table 2: Cq values of the MS2 / Mh multiplex qPCR reaction

[0044] Example 2 DNA interference test of the multiplex qPCR reagent

[0045] To detect whether human genomic DNA (gDNA) samples would interfere with the multiplex qPCR reagent and thus affect the results of the multiplex qPCR, the nucleic acid extracted from TF-1a human erythroblast cells was quantified by Qubit dsDNA and then 100 ng or 200 ng was added to the multiplex qPCR reagent, and the multiplex qPCR reaction was carried out. The judgment criterion for the interference test in this example was based on the Cq value of the multiplex qPCR reaction of the control group (without adding human genomic DNA samples). If the difference between the Cq value of the multiplex qPCR reaction of the multiplex qPCR reagent with added human genomic DNA samples and the Cq value of the control group was less than or equal to 1 cycle (i.e., ΔCq ≤ 1), it was judged as no interference; if the difference between the Cq value of the multiplex qPCR reaction of the multiplex qPCR reagent with added human genomic DNA samples and the Cq value of the control group exceeded 1 cycle (i.e., more than one cycle delay, ΔCq > 1), it was judged as interference.

[0046] Referring to Table 3, the results show that compared with the control group, the difference in the Cq value of the MS2 qPCR reaction of the multiplex qPCR reagent with the addition of 100 ng of human genomic DNA sample is less than 1 cycle, while the difference in the Cq value of the MS2 qPCR reaction of the multiplex qPCR reagent with the addition of 200 ng of human genomic DNA sample is close to 1 cycle. Therefore, it is judged that the upper limit of the tolerance of MS2 qPCR to human genomic DNA is about 200 ng gDNA.

[0047] Table 3: Cq values of the MS2 qPCR reaction of the multiplex qPCR reagent with the addition of human genomic DNA samples Cq value MS2 RNA 100 cp (control group) 33.84 MS2 RNA 100 cp + 3 μl PBS buffer 33.91 MS2 RNA 100 cp + 100 ng gDNA 34.37 MS2 RNA 100 cp + 100 ng gDNA 34.56 MS2 RNA 100 cp + 6 μl PBS buffer 33.75 MS2 RNA 100 cp + 200 ng gDNA 34.88 MS2 RNA 100 cp + 200 ng gDNA 34.89 NTC NA

[0048] Referring to Table 4, the results show that compared with the control group, there is almost no difference in the Cq value of the Mh qPCR reaction of the multiplex qPCR reagent with the addition of 100 ng or 200 ng of human genomic DNA sample. Therefore, it is judged that the upper limit of the tolerance of Mh qPCR to human genomic DNA is greater than 200 ng gDNA.

[0049] Table 4: Cq values of the Mh qPCR reaction of the multiplex qPCR reagent with the addition of human genomic DNA samples

[0050] Example 3: Using the multiplex qPCR reaction to detect the extraction recovery rate of the G1 extraction cartridge of the Dagene G1 fully automatic nucleic acid detection system and evaluate whether there are interference and inhibition factors in the G1 extraction cartridge

[0051] Referring to Figure 6 , the quality control method of the nucleic acid detection system of the present invention further includes the steps of: Step S60: Mix the quantified Escherichia coli phage MS2 into the extraction, the first sequence group, the Mycoplasma haemofelis Mh plastid, the first sequence group and the second sequence group, and perform the second multiplex real-time polymerase chain reaction to obtain the second Mh Cq value; when the difference between the first Mh Cq value and the second Mh Cq value is less than or equal to 1, there are no interference and inhibition factors in the eluate or the extraction cartridge of the nucleic acid detection system; and when the difference between the first Mh Cq value and the second Mh Cq value is greater than 1, there are interference and inhibition factors in the eluate or the extraction cartridge of the nucleic acid detection system.

[0052] Referring to Figure 7 , the quality control method of the nucleic acid detection system of the present invention further includes the steps of: Step S71: Dilute the eluate by 10 times to obtain the eluate diluted 10 times; and Step S72: Mix the undiluted eluate, the 10-fold diluted eluate, Mycoplasma haemofelis (Mh) plasmid, the first sequence group, and the second sequence group respectively, and perform multiplex real-time polymerase chain reaction to obtain the second MS2 Cq value; when the difference between the MS2 Cq value of the undiluted eluate (i.e., the first MS2 Cq value) and the MS2 Cq value of the 10-fold diluted eluate (i.e., the second MS2 Cq value) is greater than or equal to 3, or when the difference between the MS2 Cq value of the undiluted eluate (i.e., the first MS2 Cq value) and the MS2 Cq value of the 10-fold diluted eluate (i.e., the second MS2 Cq value) is less than 3, there are interference and inhibition factors in the eluate.

[0053] (1) Detect the extraction recovery rate of the G1 extraction cartridge of the Dagene G1 fully automatic nucleic acid detection system using multiplex qPCR reaction:

[0054] Mix the 10^-5 diluted Escherichia coli phage with TF-1a human erythroblast cells to prepare a simulated sample. Take 400 μl of this sample and place it into the G1 extraction cartridge (about 8.97E+03 copies of Escherichia coli phage) for nucleic acid extraction. In this example, 3 Dagene G1 fully automatic nucleic acid detection systems are used to perform nucleic acid extraction in 3 batches respectively (a total of 9 G1 extraction cartridges). After extraction, take 12 μl of the 10-fold diluted eluate and add it to the multiplex qPCR reagent containing 10^3 copies of Mh DNA plasmid for multiplex qPCR reaction, and use the Bio Rad CFX96 instrument to quantify the recovery rate of the G1 extraction cartridge.

[0055] In addition, use another commercial brand: the Dot 4810 automatic nucleic acid extractor and the direct heating lysis method for nucleic acid extraction as a control group. After quantification, calculate the recovery rate of nucleic acid extraction.

[0056] Referring to Table 5, the results show that the nucleic acid extraction recovery rate of batch 2 of machine G8 is significantly lower than that of the Dot 4810 automatic nucleic acid extractor and the direct heating lysis method. Therefore, multiplex qPCR reaction can be used to evaluate the recovery efficiency of different batches of G1 extraction cartridges.

[0057] Table 5: Nucleic acid extraction recovery rate of the G1 extraction cartridge of the Dagene G1 fully automatic nucleic acid detection system relative to the direct heating lysis method and the Dot 4810 automatic nucleic acid extractor

[0058] (2) Detect whether there are interference and inhibition factors in the G1 extraction cartridge of the Dagene G1 fully automatic nucleic acid detection system using multiplex qPCR reaction:

[0059] The criteria for judging whether there is an interference inhibitor in the G1 extraction cartridge are as follows: (a) Based on the Mh Cq value of the multiplex qPCR reaction of the control group (without adding eluent), if the difference between the Mh Cq value of the multiplex qPCR reaction of the multiplex qPCR reagent with added eluent and the Mh Cq value of the control group is less than or equal to 1 cycle (i.e., MhΔCq ≤ 1), it is judged as no interference; conversely, if the difference between the Mh Cq value of the multiplex qPCR reaction of the multiplex qPCR reagent with added eluent and the Mh Cq value of the control group is greater than 1 cycle (i.e., MhΔCq > 1), it is judged as interference; and (b) Based on the MS2 Cq value of the multiplex qPCR reaction of the control group (added with 1-fold diluted eluent), if the difference between the MS2 Cq value of the multiplex qPCR reaction of the multiplex qPCR reagent with added 10-fold diluted eluent and the MS2 Cq value of the control group is greater than or equal to 3 cycles (i.e., MS2ΔCq ≥ 3), it is judged as no interference; conversely, if the difference between the MS2 Cq value of the multiplex qPCR reaction of the multiplex qPCR reagent with added 10-fold diluted eluent and the MS2 Cq value of the control group is less than 3 cycles (i.e., MS2ΔCq < 3), it is judged as interference.

[0060] To evaluate the difference in the Mh Cq values of the multiplex qPCR reactions of the multiplex qPCR reagents with and without added eluent, as shown in Table 1 of Example 1, the average value of the Mh Cq values of the multiplex qPCR reactions of the multiplex qPCR reagent without added eluent was 28.59. In addition, Table 6 shows the Mh Cq values of the multiplex qPCR reactions of the multiplex qPCR reagents with added eluent (1-fold diluted eluent) in 3 batches using 3 Dagene G1 fully automatic nucleic acid detection systems respectively, and the difference values between each Mh Cq value and the average value (28.59) of the Mh Cq values of the multiplex qPCR reactions of the multiplex qPCR reagent without added eluent. The results show that ΔCq > 1 for Batch 2 of Machine G8 and Batch 1 of Machine G25 (as underlined), and accordingly it is judged that there is an interference inhibitor in the G1 extraction cartridges of Batch 2 of Machine G8 and Batch 1 of Machine G25.

[0061] Table 6: Mh Cq values of the multiplex qPCR reactions of the multiplex qPCR reagents with added eluent (1-fold diluted eluent) in 3 batches using 3 Dagene G1 fully automatic nucleic acid detection systems respectively, and the difference value (ΔCq) between each of them and the average value (28.59) of the Mh Cq values of the multiplex qPCR reactions of the multiplex qPCR reagent without added eluent.

[0062] To evaluate the difference in the MS2 Cq values of the multiplex qPCR reactions of the multiplex qPCR reagents added with eluents diluted 10-fold and 1-fold, Table 7 shows the MS2 Cq values of the multiplex qPCR reactions of the multiplex qPCR reagents added with eluents diluted 10-fold and 1-fold in 3 batches respectively using 3 Dagene G1 fully automatic nucleic acid detection systems, as well as the difference values between them. The results show that ΔCq of Batch 2 and Batch 3 of Machine G8, Batch 1 and Batch 3 of Machine G9, and Batch 1 and Batch 3 of Machine G25 < 3. Based on this, it is judged that there are interference and inhibition factors in the G1 extraction cartridges of Batch 2 and Batch 3 of Machine G8, Batch 1 and Batch 3 of Machine G9, and Batch 1 and Batch 3 of Machine G25.

[0063] Table 7: The MS2 Cq values of the multiplex qPCR reactions of the multiplex qPCR reagents added with eluents diluted 10-fold and 1-fold in 3 batches respectively using 3 Dagene G1 fully automatic nucleic acid detection systems, as well as the difference values between them.

[0064] As can be seen from the above, the acceptance criteria for the interference and inhibition factor test of the eluent in the G1 extraction cartridge of the Dagene G1 fully automatic nucleic acid detection system are as follows: the Mh Cq value of the eluent diluted 1-fold and the Mh Cq value without the eluent are less than or equal to 1 cycle (i.e., MhΔCq ≤ 1), and the difference value between the MS2 Cq values of the multiplex qPCR reactions of the eluent diluted 1-fold and the eluent diluted 10-fold is greater than or equal to 3 (MS2ΔCq ≥ 3).

[0065] Example 4 Instrument quality control of the Dagene G1 fully automatic nucleic acid detection system using multiplex qPCR reaction

[0066] See Figure 8 , the quality control method of the nucleic acid detection system of the present invention further includes the steps of: Step S81: Repeat the steps of Step S10 to Step S40 multiple times to obtain multiple MS2 Cq values and multiple Mh Cq values, and calculate the average value and standard deviation of the multiple MS2 Cq values, as well as the average value and standard deviation of the multiple Mh Cq values; wherein the standard range of the quality control of the nucleic acid detection system includes the average value of the MS2 Cq values + / - 2 x the standard deviation of the MS2 Cq values, and the average value of the Mh Cq values + / - 2 x the standard deviation of the Mh Cq values; and Step S82: When the obtained MS2 Cq value or Mh Cq value is not within the standard range, it is determined that the detection fails to pass the quality control standard.

[0067] A 10^-5 diluted E. coli phage MS2 and 4x 10^4 TF-1a human erythrocyte progenitor cells were mixed and placed in a G1 extraction cartridge for nucleic acid extraction. In this embodiment, 4 Dagene G1 fully automatic nucleic acid detection systems were used to extract nucleic acids (a total of 22 G1 extraction cartridges, each G1 extraction cartridge performed 6 reactions, and a total of 132 results were obtained). After the G1 cartridge extraction, 12μl of the diluted eluate was directly injected into the multiplex qPCR reagent containing 10^3 copies of the Mh DNA plasmid to perform a multiplex qPCR reaction.

[0068] In addition, nucleic acid extraction was performed using the existing Dot 4810 automated nucleic acid extractor to obtain an eluate, and 12 μl of the eluate was added to a multiplex qPCR reagent containing 10^3 copies of Mh DNA plasmids. Multiplex qPCR reactions were performed using the Dagene G1 fully automated nucleic acid detection system (a total of 42 results were obtained) as a control group to confirm that there were no problems with the specimens used in the entire extraction process and the G1 qPCR process.

[0069] The instrument quality control test results were statistically analyzed and abnormal data were excluded to obtain the average results of MS2 Cq values and Mh Cq values for nucleic acid extraction and multiple qPCR reactions using the DageneG1 fully automatic nucleic acid detection system as shown in Table 8. Based on these results, it is estimated that the reasonable quality control standard range can be set as: the average value of MS2 Cq value + / -2x standard deviation (SD), and the average value of Mh Cq value + / -2x SD.

[0070] Table 8: The average results of MS2 Cq value and Mh Cq value of nucleic acid extraction and multiple qPCR reaction using Dagene G1 fully automatic nucleic acid detection system, and the average results of MS2 Cq value and Mh Cq value of the control group using Dot 4810 automated nucleic acid extraction instrument combined with G1 qPCR.

[0071] In addition, referring to Table 9, the results of the automated nucleic acid extractor Dot 4810 combined with the G1 qPCR show that the Cq value of the sixth reaction tank MS2 of the G25 machine is abnormal, exceeding the range of the average value of the MS2 Cq value + / - 2x standard deviation; the Cq values of the fifth and sixth reaction tanks Mh of the G25 machine are abnormal, and the Mh signal appears outside the range of the average value of the Mh Cq value + / - 2x standard deviation. The probability of this abnormal result is 50% (one abnormal result appears in two experiments). The abnormal values are shown as the bottom line, indicating that there are abnormal factors in the qPCR system of the G25 machine.

[0072] Referring to Table 10, the abnormalities in the fifth and sixth reaction tanks of the G25 machine are also shown in the full-process results of the Dagene G1 fully automated nucleic acid detection system. Abnormal values of the MS2 signal exceeding the range of the average value of the MS2 Cq value + / - 2x standard deviation, and abnormal values of the Mh signal exceeding the range of the average value of the Mh Cq value + / - 2x standard deviation (as shown by the bottom line) appear. Subsequently, the G25 machine was inspected. The results showed that there were foreign objects in the holes of the heating seats and the holes of the LED filters in the fifth and sixth reaction tanks of the G25 machine, resulting in problems with the heating and fluorescence signal reception of qPCR. Therefore, multiplex qPCR reactions can detect whether there are abnormalities in the Dagene G1 fully automated nucleic acid detection system.

[0073] Table 9: Using the automated nucleic acid extractor Dot 4810 combined with the Dagene G1 fully automated nucleic acid detection system to identify abnormal machines.

[0074] Table 10: Detection results of the instrument quality control of the full process of the abnormal Dagene G1 fully automated nucleic acid detection system

[0075] Example 4 Using multiplex qPCR reactions for quality control of the G1 extraction cartridge of the Dagene G1 fully automated nucleic acid detection system

[0076] A mixture of 10^5 diluted Escherichia coli phage MS2 and 4x10^4 TF-1a human erythroblast cells was put into an expired G1 extraction cartridge for nucleic acid extraction. It is known that expired cartridges will affect the overall nucleic acid recovery rate. In this example, the Dagene G1 fully automated nucleic acid detection system was used for six nucleic acid extractions (a total of 6 results). After extraction, 12 μl of the eluate was automatically injected into the multiplex qPCR reagent containing 10^3 copies of the Mh DNA plasmid in the lower cartridge for multiplex qPCR reactions.

[0077] Table 11 shows the test results of the quality control of the G1 extraction cartridge of the Dagene G1 fully automated nucleic acid detection system. The results show that in the first reaction well, the second reaction well, the third reaction well and the sixth reaction well, abnormal values of the MS2 signal exceeding the range of the average value of the MS2 Cq value + / - 2x standard deviation, and abnormal values of the Mh signal exceeding the range of the average value of the Mh Cq value + / - 2x standard deviation (as shown by the bottom line). Therefore, the multiplex qPCR reaction can detect whether the extraction recovery rate of the G1 extraction cartridge of the Dagene G1 fully automated nucleic acid detection system is abnormal.

[0078] Table 10: Test Results of the Quality Control of the G1 Extraction Cartridge of the Dagene G1 Fully Automated Nucleic Acid Detection System MS2 Cq value Mh Cq value First reaction tank <![CDATA 28.23 > <![CDATA 29.05 > Second reaction tank <![CDATA 28.09 > 28.14 Third reaction tank <![CDATA 28.36 > 28.33 Fourth reaction tank 28.02 27.87 Fifth reaction tank 27.98 27.81 Sixth reaction tank <![CDATA 28.34 > 28.12 Average value 28.17 28.22 SD 0.16 0.45 CV (%) 0.58 1.59

[0079] The advantages of the present invention are as follows:

[0080] High specificity: The MS2 forward primer shown in SEQ ID NO:1, the MS2 reverse primer shown in SEQ ID NO:2, and the MS2 probe shown in SEQ ID NO:3 from Escherichia coli phage MS2, and the Mh forward primer shown in SEQ ID NO:4, the Mh reverse primer shown in SEQ ID NO:5, and the Mh probe shown in SEQ ID NO:6 from Mycoplasma haemofelis of the quality control kit of the present invention have high specificity and cannot recognize the sequences of other species. MS2 phage mainly exists in contaminated wastewater. The designed primers and probes are developed and designed based on bioinformatics analysis by widely comparing the NCBI Database sequences. The Primer-Blast results will not recognize the sequences of other species. The paired primers and probes of Mycoplasma haemofelis, whose pathogen is mainly pets, also have good specificity in the Primer-Blast analysis and will not recognize other sequences. These two species do not exist in the experimental environment and reagent formulations, nor do they belong to human pathogen nucleic acid detection products. Therefore, they can be designed for use in the factory quality control process. When actually applied to the quality control of the G1 fully automated nucleic acid detection system, no non-specific signals will be generated.

[0081] Multifunction and applications: The MS2 forward primer shown in SEQ ID NO:1, the MS2 reverse primer shown in SEQ ID NO:2, and the MS2 probe shown in SEQ ID NO:3 can be used as standards for nucleic acid extraction in the G1 extraction cartridge of the Dagene G1 fully automated nucleic acid detection system to evaluate the recovery rate of nucleic acid extraction; and the Mh forward primer shown in SEQ ID NO:4, the Mh reverse primer shown in SEQ ID NO:5, and the Mh probe shown in SEQ ID NO:6 can be used to evaluate the presence of interference and inhibitory factors in the G1 extraction cartridge and as an endogenous control for qPCR reagents. Therefore, through multiplex qPCR reactions, this quality control kit can detect the nucleic acid extraction recovery rate of the G1 extraction cartridge of the Dagene G1 fully automated nucleic acid detection system, evaluate whether there are interference and inhibitory factors in the eluate obtained after nucleic acid extraction of the sample, the quality control of the extraction cartridge of this nucleic acid detection system, or the quality control of the instrument of this nucleic acid detection system.

[0082] Not easily interfered by the nucleic acid concentration of the human genome: The upper tolerance limit of human genomic DNA in this multiplex qPCR reaction is 200 ng. Therefore, when this quality control kit is used to detect samples from the human upper respiratory tract, the qPCR reaction is not easily affected by the sample concentration.

[0083] High sensitivity: For 10 copies of MS2 RNA / rxn in the G1 fully automated nucleic acid detection system and the Bio Rad system, the detection rates are both 100%. For extraction systems with poor recovery rates, the recovery rates can be estimated.

[0084] Although the present invention has disclosed preferred embodiments, it is not intended to limit the present invention. Any person of ordinary skill in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be defined by the appended patent application scope. Description of reference numerals

[0085] S10: Step S20: Step S30: Step S40: Step S50: Step S60: Step S71: Step S72: Step S81: Step S82: Step.

Claims

1. A quality control kit for a nucleic acid detection system, comprising Escherichia coli phage MS2 and a first sequence group, and the first sequence group includes: A forward primer having the sequence shown in SEQ IQ NO:1; A reverse primer having the sequence shown in SEQ IQ NO:2; And A probe having the sequence shown in SEQ IQ NO:

3.

2. The quality control kit for a nucleic acid detection system according to claim 1, further comprising Mycoplasma haemofelis Mh plastid and a second sequence group, and the second sequence group includes: A forward primer having the sequence shown in SEQ IQ NO:4; A reverse primer having the sequence shown in SEQ IQ NO:5; And A probe having the sequence shown in SEQ IQ NO:

6.

3. A quality control method for a nucleic acid detection system, comprising the steps of: Step S10: Provide the quality control kit for the nucleic acid detection system according to claim 2; Step S20: Provide a sample; Step S30: Mix the sample with the Escherichia coli phage MS2 and put it into the extraction cartridge of the nucleic acid detection system for nucleic acid extraction to obtain an eluate; Step S40: Mix the eluate, the Mycoplasma haemofelis Mh plastid, the first sequence group and the second sequence group, and perform a first multiplex real-time polymerase chain reaction to obtain a first MS2 Cq value and a first Mh Cq value; and Step S50: Determine the recovery efficiency of nucleic acid extraction according to the first MS2 Cq value.

4. The quality control method for a nucleic acid detection system according to claim 3, further comprising the steps of: Step S60: Put the quantified Escherichia coli phage MS2 into extraction, mix the first sequence group, the Mycoplasma haemofelis Mh plastid, the first sequence group and the second sequence group, and perform a second multiplex real-time polymerase chain reaction to obtain a second Mh Cq value; when the difference between the first Mh Cq value and the second Mh Cq value is less than or equal to 1, there is no interference inhibitor in the eluate or the extraction cartridge of the nucleic acid detection system; and when the difference between the first Mh Cq value and the second Mh Cq value is greater than 1, there is the interference inhibitor in the eluate or the extraction cartridge of the nucleic acid detection system.

5. The quality control method for a nucleic acid detection system according to claim 3, further comprising the steps of: Step S71: Dilute the eluate by 10 times to obtain a 10-fold diluted eluate; and Step S72: Mix the 10-fold diluted eluate, the Mycoplasma haemofelis Mh plastid, the first sequence group and the second sequence group, and perform a third multiplex real-time polymerase chain reaction to obtain a second MS2 Cq value; when the difference between the second MS2 Cq value and the first MS2 Cq value is greater than or equal to 3, there is no interference inhibitor in the eluate or the extraction cartridge of the nucleic acid detection system; and when the difference between the second MS2 Cq value and the first MS2 Cq value is less than 3, there is the interference inhibitor in the eluate or the extraction cartridge of the nucleic acid detection system.

6. The quality control method of the nucleic acid detection system according to claim 4 or 5, wherein the interference inhibitor includes the inhibitor in the eluent.

7. The quality control method of the nucleic acid detection system according to claim 3, wherein the upper limit of the DNA concentration of the specimen is 200 ng.

8. The quality control method of the nucleic acid detection system according to claim 3, further comprising the steps of: Step S81: Repeating the steps as described in claim 3 multiple times to obtain multiple MS2 Cq values and multiple Mh Cq values, and calculating the average value and standard deviation of the multiple MS2 Cq values, and the average value and standard deviation of the multiple Mh Cq values; wherein the standard range of the quality control of the nucleic acid detection system includes the average value of the MS2 Cq values + / - 2 x the standard deviation of the MS2 Cq values, and the average value of the Mh Cq values + / - 2 x the standard deviation of the Mh Cq values; and Step S82: When the obtained MS2 Cq value or Mh Cq value is not within the standard range, it is determined that the detection fails to pass the quality control standard.

9. The quality control method of the nucleic acid detection system according to claim 3, wherein the nucleic acid detection system includes a fully automatic nucleic acid detection system.

10. The use of a quality control kit for the nucleic acid detection system according to claim 1 or 2, wherein the quality control kit for the nucleic acid detection system is used to evaluate the recovery rate of the specimen after nucleic acid extraction, whether there is residual interference inhibitor in the eluent obtained after nucleic acid extraction of the specimen, the quality control of the extraction cartridge of the nucleic acid detection system, or the quality control of the instrument of the nucleic acid detection system.

Images