阅读说明：本技术 一种检测核酸质谱设备性能的方法 (Method for detecting performance of nucleic acid mass spectrometry equipment ) 是由 相双红 郭惠民 朱成顺 于 2021-02-05 设计创作，主要内容包括：本发明涉及质谱检测技术领域,公开了一种检测核酸质谱设备性能的方法,包括步骤1)：采用合成引物稀释液制备模拟核酸样本核苷酸位点的参考品；步骤2)：编辑核酸质谱设备的参数,将参考品放入核酸质谱设备进行检测；步骤3)：检测结果得到包含核苷酸、基因型信息,并对所得信息进行分析,用于检测核酸质谱设备的性能；所述分析包含核苷酸准确度、基因型准确度、浓度响应曲线。本发明通过使用高纯度、多浓度、多分子量的合成引物的稀释液模拟核酸样本基因位点制备参考品进行质谱检测,并能根据样本参数确定核酸质谱设备的应用特性。(The invention relates to the technical field of mass spectrometry detection, and discloses a method for detecting the performance of nucleic acid mass spectrometry equipment, which comprises the following steps of 1): preparing a reference substance for simulating nucleotide sites of the nucleic acid sample by adopting a synthetic primer diluent; step 2): editing parameters of the nucleic acid mass spectrometry equipment, and placing a reference substance into the nucleic acid mass spectrometry equipment for detection; step 3): the detection result obtains information containing nucleotide and genotype, and the obtained information is analyzed for detecting the performance of the nucleic acid mass spectrometry equipment; the analysis included nucleotide accuracy, genotype accuracy, concentration response curves. The invention simulates the gene locus of the nucleic acid sample by using the diluent of the synthetic primer with high purity, multiple concentrations and multiple molecular weights to prepare the reference substance for mass spectrum detection, and can determine the application characteristics of the nucleic acid mass spectrum equipment according to sample parameters.)

1. A method of detecting the performance of a nucleic acid mass spectrometry apparatus, comprising the steps of:

step 1): preparing a reference substance for simulating nucleotide sites of the nucleic acid sample by adopting a synthetic primer diluent;

step 2): editing parameters of the nucleic acid mass spectrometry equipment, and placing a reference substance into the nucleic acid mass spectrometry equipment for detection;

step 3): the detection result obtains information containing nucleotide and genotype, and the obtained information is analyzed for detecting the performance of the nucleic acid mass spectrometry equipment; the analysis included nucleotide accuracy, genotype accuracy, concentration response curves.

2. The method according to claim 1, wherein step 1) comprises in particular:

1.1) selecting a plurality of nucleotide sites with different molecular weights within the mass range of the nucleic acid mass spectrum; each nucleotide site is designed to contain the expression of a nucleotide site consisting of 5 synthetic primers; wherein 1 primer mimics an extension primer and 4 mimics extension products A, T, C and G, respectively;

1.2) the molecular weights of the basic bases A, C, G and T of the known synthetic primers are 313.21Da, 289.18Da, 329.21Da and 304.19Da respectively; according to each nucleotide site to be detected and the molecular weight position of the nucleic acid mass spectrum, firstly determining the molecular weight m of the extension primer simulating the nucleotide; determining the molecular weight of the extension product to be simulated according to the site type of SNP mutation detection simulation and the difference value of the molecular weights of different bases;

1.3) performing a trial and error procedure by means of the formula Mw ═ An 313.21+ Cn ═ 289.18+ Gn × 329.21+ Tn × 304.19-61.94, wherein An, Cn, Gn and Tn represent the number of A, C, G and T in the sequence, respectively; enabling the difference value between the molecular weight of An extension primer or An extension product which needs to be simulated and the molecular weight of Mw to be smaller than 3Da, wherein An, Cn, Gn and Tn are integers, and enabling (Cn + Gn)/(An + Cn + Gn + Tn) to be in the range of 20-80%, and further respectively obtaining the simulated molecular weight of the extension product, the extension product A, C, G and the extension product T;

1.4) randomly arranging 5 sequences with known specific A, C, G and T base number and molecular weight in each locus to obtain 5 simulated sequences corresponding to each locus, and synthesizing and purifying to obtain synthetic primers;

1.5) respectively determining the nucleic acid mass spectra corresponding to the molecular weight in the mass range to estimate the low concentration and the high concentration; based on the number of selected nucleotide sites, the extension primer mimicked at each site is set at a low concentration;

1.6) carrying out pH value detection on the synthetic primer diluent with determined concentration, wherein the pH value of each synthetic primer diluent is within a specified range, and obtaining a reference substance simulating nucleotide sites of a nucleic acid sample.

3. The method according to claim 1 or 2, wherein in step 1): the pH value of the synthetic primer diluent is 4-10.

4. The method according to claim 1 or 2, wherein in step 1): the purity of the synthetic primer is not lower than 99%; and/or the content range of the CG basic group of the synthetic primer is 20-80%.

5. The method according to claim 2, characterized in that in step 1.1):

the number of the nucleotide sites is 1-20; and/or

The molecular weights of the plurality of nucleotide sites uniformly or non-uniformly cover the mass range of the nucleic acid mass spectrum, and the molecular weights of the synthetic primers corresponding to the adjacent nucleotide sites are not overlapped.

6. The method of claim 2, wherein in step 1.5):

according to a quadratic polynomial y-Ax²Determining equation coefficients through three-point test concentration to obtain nucleic acid mass spectrum estimation low concentration corresponding to molecular weight in a mass range; wherein x is the molecular weight and y is the estimated concentration at which the molecular weight is lowest;

concentration kinetic of nucleic acid mass spectrumThe state range is selected to be not higher than 10³The double low concentration is taken as the high concentration corresponding to the molecular weight in the mass spectrum range.

7. The method of claim 2, wherein in step 1.5), the high and low concentrations of the extension product are staggered within a nucleotide site.

8. The method of claim 1, wherein in step 2), the number n of reference sample applications is calculated before detection according to the nucleotide detection accuracy and a single set of target value minimum sample size methods.

9. The method of claim 8, wherein the number n of samples on the reference sample is calculated by the formula:wherein a is significance, p₀Is the target value.

10. The method as claimed in claim 1, wherein in step 3), the validity of the low concentration curve and the high concentration curve of the sample can be accurately detected by the nucleic acid mass spectrometry according to the peak height, the signal to noise ratio and the resolution under the condition of the concentration of the reference substance primer so as to determine the concentration response curve of the nucleic acid mass spectrometry detection device.

Technical Field

The invention relates to the technical field of mass spectrometry detection, in particular to a method for detecting the performance of nucleic acid mass spectrometry equipment.

Background

Nucleic acid mass spectrometry is an omics technology developed in recent years and used in the field of life science and technology, and has the performance advantages of good sensitivity, resolution, signal-to-noise ratio and the like, the operation advantages of rapidness, easiness in automation and the like, and the platform advantages of economy, high efficiency, medium throughput and the like, so that the nucleic acid mass spectrometry is widely applied to different fields of all over the world, such as cancer analysis, genetic disease detection, pharmacogenomics, agricultural genomics, clinical research and the like of solid tumors and liquid living tissue specimens.

The principle of nucleic acid mass spectrometry is to irradiate a cocrystal formed by a sample and a matrix by using laser and ionize the cocrystal, the ionized cocrystal film crystal can generate ions with different mass-to-nuclear ratios due to energy absorption, then the molecular weights of different types of ions in the sample are measured by a mass analyzer and are sequentially arranged from small to large to obtain a mass spectrum, the ions are detected according to different flight times of the different ions reaching a detector, namely the ions are analyzed by the fact that the mass-to-charge ratio (M/Z) of the ions is in direct proportion to the flight time of the ions, and the molecular weight of the sample is obtained.

However, in practical applications, although the nucleic acid mass spectrometer has a standard for calibrating and debugging the nucleic acid mass spectrometer during the installation and the periodic maintenance, the application evaluation of the nucleic acid mass spectrometer on the final characteristics of sample detection cannot be effectively reflected on the response of the nucleic acid mass spectrometer to the sample concentration within the full mass range. Therefore, there is a need for a standard substance that can simulate real samples for nucleotide detection accuracy and genotyping accuracy, so as to ensure the reliability and accuracy of nucleic acid mass spectrometry in practical applications.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for detecting the performance of nucleic acid mass spectrometry equipment, and particularly relates to a method for preparing a reference substance by simulating a nucleic acid sample gene locus by using a diluent of a high-purity, multi-concentration and multi-molecular-weight synthetic primer to carry out mass spectrometry detection, and the application characteristics of the nucleic acid mass spectrometry equipment can be determined according to sample parameters.

The specific technical scheme of the invention is as follows: a method of detecting the performance of a nucleic acid mass spectrometry apparatus comprising the steps of:

step 1): and preparing a reference substance for simulating the nucleotide sites of the nucleic acid sample by adopting the synthetic primer diluent.

Step 2): editing the parameters of the nucleic acid mass spectrometry equipment, and placing the reference substance into the nucleic acid mass spectrometry equipment for detection.

Step 3): the detection result obtains information containing nucleotide and genotype, and the obtained information is analyzed for detecting the performance of the nucleic acid mass spectrometry equipment; the analysis includes but is not limited to nucleotide accuracy, genotype accuracy, concentration response curve.

Preferably, step 1) specifically comprises:

1.1) selecting a plurality of nucleotide sites with different molecular weights within the mass range of the nucleic acid mass spectrum according to the nucleic acid mass spectrum detection principle; each nucleotide site is designed to contain the expression of a nucleotide site consisting of 5 synthetic primers; wherein 1 primer simulates extension primer, 4 primers simulate extension products A, T, C and G respectively, and the molecular weight difference is shown in tables 1-2:

table 1: single base T-type extension

Table 2: single base U-shaped extension

1.2) the molecular weights of the basic bases A, C, G and T of the known synthetic primers (oligonucleotides) are 313.21Da, 289.18Da, 329.21Da and 304.19Da, respectively; according to each nucleotide site to be detected and the molecular weight position of the nucleic acid mass spectrum, firstly determining the molecular weight m of the extension primer simulating the nucleotide; and determining the molecular weight of the extension product to be simulated according to the site type simulated by the detection of the SNP mutation and the molecular weight difference (table 1 or table 2) of different bases.

1.3) performing a trial and error procedure by means of the formula Mw ═ An 313.21+ Cn ═ 289.18+ Gn × 329.21+ Tn × 304.19-61.94, wherein An, Cn, Gn and Tn represent the number of A, C, G and T in the sequence, respectively; the differences between An, Cn, Gn and Tn which are integers and Mw and the molecular weight of the determined extension primer or extension product to be simulated are smaller than 3Da, and the molecular weight (Cn + Gn)/(An + Cn + Gn + Tn) is within the range of 20-80%, so that the simulated molecular weights of the extension product, the extension product A, C, G and T are obtained respectively.

Because the minimum molecular weight difference is 9Da, the mass-to-charge ratio of each synthesized primer mass spectrum peak needs to be distinguished, and the primers can be accurately identified only when the primers need to be analyzed by internal error software of at least 3 Da; the higher the GC content of A, C, G, T in nucleic acid, the higher the DNA density, and the less heat and alkali can denature the DNA, the too high or too low CG content is not beneficial to the ion formation in the nucleic acid mass spectrum detection, and the CG content is controlled to 20-80% when the synthetic primer is made, so that the actual sample can be effectively excited and simulated.

1.4) randomly arranging the 5 sequences with known specific A, C, G and T base number and molecular weight in each locus to obtain 5 simulated sequences corresponding to each locus, synthesizing and purifying to obtain the synthetic primers.

1.5) respectively determining the nucleic acid mass spectra corresponding to the molecular weight in the mass range to estimate the low concentration and the high concentration; depending on the number of nucleotide sites selected, the extension primer mimicked at each site is set at a low concentration.

1.6) carrying out pH value detection on the synthetic primer diluent with determined concentration, wherein the pH value of each synthetic primer diluent is within a specified range, and obtaining a reference substance simulating nucleotide sites of a nucleic acid sample.

Preferably, in step 1): the pH value of the synthetic primer diluent is 4-10. Preferably, the pH is 7.

Preferably, in step 1): the purity of the synthesized primer is not less than 99%.

Preferably, the synthetic primer has a CG base content ranging from 20% to 80%.

Preferably, in step 1.1): the number of the nucleotide sites is 1-20. The molecular weights of the plurality of nucleotide sites uniformly or non-uniformly cover the mass range of the nucleic acid mass spectrum, and the molecular weights of the synthetic primers corresponding to the adjacent nucleotide sites are not overlapped.

Preferably, in step 1.2), the molecular weight m of the extension primer and the molecular weight m + 247.2-m +287.2 of the extension product (T-type extension) or m + 247.2-m +327.2 (U-type extension) need to satisfy the mass range of the nucleic acid mass spectrum; the molecular weight of the four extension products corresponding to the extension primers is in the mass range of the nucleic acid mass spectrum.

Preferably, in step 1.5): according to a quadratic polynomial y-Ax²Determining equation coefficients through three-point test concentration to obtain nucleic acid mass spectrum estimation low concentration corresponding to molecular weight in a mass range; wherein x is the molecular weight and y is the estimated concentration at which the molecular weight is lowest; the dynamic range of concentration of nucleic acid mass spectrum is not higher than 10³The double low concentration is taken as the high concentration corresponding to the molecular weight in the mass spectrum range.

Preferably, in step 1.5), the high and low concentrations of the extension product are staggered within the nucleotide position.

According to the setting of different high and low concentration sites, the mass spectrum detection data of high concentration or low concentration can be connected to obtain mass spectrum concentration response curves of high concentration or low concentration respectively.

Preferably, in step 2), the number n of reference sample loads is calculated before detection according to the nucleotide detection precision and a single set of target value (OPC) minimum sample size method.

Preferably, the calculation formula of the sample loading number n of the reference sample is as follows:wherein a is significance, p₀Is the target value.

Preferably, in step 3), the validity of a low concentration curve and a high concentration curve of a sample can be accurately detected by nucleic acid mass spectrometry according to the peak height, the signal-to-noise ratio and the resolution ratio under the condition of the concentration of the reference primer, so as to determine the concentration response curve of the nucleic acid mass spectrometry detection device.

Compared with the prior art, the invention has the beneficial effects that: the invention simulates the gene locus of the nucleic acid sample by using the diluent of the synthetic primer with high purity, multiple concentrations and multiple molecular weights to prepare the reference substance for mass spectrum detection, and can determine the application characteristics of the nucleic acid mass spectrum equipment according to sample parameters.

Drawings

FIG. 1 is a nucleic acid mass spectrum of a mock nucleic acid site of the synthetic primer of the example;

FIG. 2 is a mass spectrum of a reference substance in the examples.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1

According to the nucleic acid mass spectrum detection principle, a plurality of nucleotide sites with uniform or non-uniform molecular weights covering the mass range of the nucleic acid mass spectrum are selected in the mass range of the nucleic acid mass spectrum. Each nucleotide site was designed to contain the expression of 5 synthetic primer nucleotide sites (the molecular weights of the synthetic primers for adjacent nucleotide sites do not overlap). The molecular weight differences of 1 primer mimic extension primer and 4 mimic extension products (A, T, C, G) are shown in tables 1-2.

Table 1: single base T-type extension

Table 2: single base U-shaped extension

The molecular weights of the basic bases A, C, G and T of the synthetic primers (oligonucleotides) are known to be 313.21Da, 289.18Da, 329.21Da, 304.19Da, respectively. The nucleic acid of a living body is an arrangement of A, C, G and T base types, and when detecting SNP sites, a detected base is generally placed at two ends of a nucleic acid sequence, if the site has a mutation, for example, the base A of the A sequence is mutated into the base T of the T sequence, and the molecular weights of the two sequences are different from 9 Da; a. The molecular weight difference can be detected by nucleic acid mass spectrometry, and then the mutation of the SNP site is detected. According to the detection site and the detection position of the molecular weight of the nucleic acid mass spectrum, firstly, the molecular weight m of the extension primer of the analog nucleotide, the molecular weight span m + 247.2-m +287.2 (T-type extension) or the molecular weight span m + 247.2-m +327.2 (U-type extension) of the extension product of the analog nucleotide are determined to meet the mass range of the nucleic acid mass spectrum, the small difference of the molecular weight of the T-type extension belongs to the inherent base type of a living body, and the large difference of the molecular weight of the U-type extension is through the modified base, thereby being more beneficial to the identification of the nucleic acid mass. And determining the molecular weight of the extension product to be simulated according to the site type simulated by the detection of the SNP mutation and the table 1 or the table 2. By the following formula:

Mw＝An*314.21+Cn*289.18+Gn*329.21+Tn*304.19-61.94

and performing trial and error to ensure that An, Cn, Gn and Tn are integers, the difference between Mw and the determined molecular weight is less than 3Da, and the (Cn + Gn)/(An + Cn + Gn + Tn) is within the range of 20-80%.

Preferred as examples: the molecular weight of the mimic sequence is 4007.69, the mimic sequence is an oligonucleotide sequence of 'AAAAATTCCGGGG', the molecular weight of the mimic sequence is 4007.69, the molecular weight of the mimic sequence is shown in Table 3 after extension, the sequence of A, T, C, G of the oligonucleotide (primer) in the 2 nd to 5 th rows in the Table 3 is obtained through a trial and error method, the molecular weight and the molecular weight difference can be obtained, and when the nucleic acid mass spectrum is detected, the molecular weight of the oligonucleotide is allowed to have 3Da deviation, so the mimic oligonucleotide can be detected as the extension product when the nucleic acid mass spectrum is detected.

Table 3: trial and error molecular scale

The 5 bases obtained containing specific ATCG were randomized to obtain 5 synthetic primer sequences, and oligonucleotides (primers) were directly synthesized based on the randomized primer sequences, as shown in Table 4.

Table 4: random arrangement of synthetic primer sequences

Base random sequence	Molecular weight	Length of base	Description of the invention
				GATCTCAAGAAGG	4007.69	13	Extension primer
CGGCCTGTTACAGG	4279.82	14	Mimotol A base
				CCAACTTAGATAAG	4255.85	14	Mimic C base
CTAACAGGATTAAG	4295.88	14	Mimic G base
				AGCTAATTAGAGAG	4335.91	14	Mimic U base

A nucleic acid mass spectrum of the nucleic acid sites is simulated by synthesizing primers as shown in FIG. 1.

According to the method, the nucleotide which is arbitrarily set in a simulated mode in the mass range of the nucleic acid mass spectrum can be obtained.

Preferably, the nucleotide sequence contains 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, etc., and the number of the set nucleotides is determined by the accuracy of nucleotide detection. Preferably, as an example, 15 nucleotides are set, each nucleotide has 5 synthetic primers, and 75 synthetic primers in total are set, as shown in Table 5.

Table 5: 15 site synthesis primer sequences and site correspondence table

The coverage range of the molecular weight of the reference substance is 4000-10000 Da, the primers can be synthesized according to the primer sequences on the table and purified by adopting an HPLC (high performance liquid chromatography) mode, and the purity of the synthesized primers is ensured to be not less than 99%.

When nucleic acid mass spectrometry is carried out, the peak height requires that the sample concentration is increased along with the increase of molecular weight under the same condition, the minimum concentration is also increased, and the minimum concentration approximately follows quadratic polynomial y which is Ax²+ Bx + C (x is the molecular weight, y is the lowest estimated concentration of the molecular weight), and determining equation coefficients through three-point test concentrations to obtain the estimated low concentration of the nucleic acid mass spectrum corresponding to the molecular weight in the mass range; the dynamic range of concentration of nucleic acid mass spectrum is not higher than 10³The concentration is taken as the high concentration in the mass spectrum range; according to the number of the selected nucleotide sites, the extension primer simulated at each site is set according to the low concentration, and the high concentration and the low concentration of the extension product are preferably staggered in the nucleotide sites, so that the application characteristics of the nucleic acid mass spectrum can be detected.

By way of example, aiming at the performance confirmation of a DP-TOF type time-of-flight mass spectrometry detection system, the equation coefficients of a quadratic polynomial are determined by the above concentrations as follows: a-1.736E-08, B-1.351E-04, C-0.4073; with a concentration response dynamic range of 10²The concentration of each primer is shown in Table 6, wherein the nucleotide column indicates the type of nucleotide that can be simulated by the reference, the genotype column indicates the type of genotype that can be simulated by the reference, the low concentration indicates that the concentration of the corresponding synthetic primer is calculated according to the low concentration, and the high concentration indicates that the concentration of the corresponding synthetic primer is calculated according to the low concentration, wherein P in the simulated base sequence indicates that the primer is an extension primer, all calculated according to the lowest concentration.

Table 6: 15 sites 75 synthetic primer concentration and performance parameter table

And (3) detecting the pH value of the diluent with the determined synthetic primer concentration, wherein the pH value of each primer diluent is in a range of 4-10 (in the embodiment, the pH value is 7), mixing the primers according to a proportion, adding 0.1% tween, and uniformly mixing to obtain a reference substance of the simulated sample.

Calculating the sample loading quantity of the reference sample according to the nucleotide detection precision and a single group target value (OPC) minimum sample quantity method,wherein a is significant, p₀Is the target value. Let a equal to 0.05, P₀And when the content is 99.9%, n is 2994 nucleotides. Since each reference contains 4 × 15-60 nucleotides, the loading well is 2994/60 ≈ 50 holes. Under the condition that the confidence probability of the detection result is 95%, the lower confidence limit of the nucleotide detection is 99.90%, the upper confidence limit is 99.93%, and the confidence interval is 0.03%, so that the requirement on the precision of the high-precision nucleotide detection is met.

And (3) carrying out 50-hole spotting according to the requirement of nucleic acid mass spectrum detection, and detecting to obtain mass spectrum data of a reference substance, as shown in figure 2.

According to the fact that each locus contains 1 genotype, the accuracy and the confidence coefficient of the genotype in the detection result can be obtained according to the number of the loci and the number of the sample loading holes. Taking the above reference product test as an example, 15 × 50 — 750 genotypes are detected in total, the lower confidence limit of the genotype detection result is 99.60%, the upper confidence limit is 99.73%, and the confidence interval is 0.13% under the condition that the confidence probability is 95%, and the requirement on the precision of high-precision nucleotide detection is met.

When the detection precision requirement changes, the number of the sample loading holes can be adjusted according to the method, so that the requirement of detecting the detection characteristic of the nucleic acid mass spectrum is met.

After the two main indexes are detected, the effectiveness of a low-concentration curve and a high-concentration curve of a sample can be accurately detected by nucleic acid mass spectrometry according to the peak height, the signal-to-noise ratio and the resolution ratio of a reference substance primer under the concentration condition, so that the concentration response curve of the nucleic acid mass spectrometry detection equipment is determined. Preferably, taking the above reference as an example, the peak heights of all mass spectra are not less than 5, the signal-to-noise ratio is not less than 10, and the resolution is greater than the molecular weight/16, which indicates that the nucleic acid mass spectrum meets the detection of low-concentration and high-concentration samples determined by the reference within the mass range, and a concentration curve is drawn according to the concentration of each synthesized primer in the reference, that is, the detection sample of the nucleic acid mass spectrum detection device at least meets the calibration range of the concentration curve.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.