G6PD deficiency screening kit and application

Abstract

The invention provides a G6PD deficiency screening kit and application, and relates to the technical field of molecular diagnosis. The kit provided by the invention comprises a primer and a sample treatment solution, wherein the sample treatment solution is prepared from 2.0 to 5.0 mM of Mg < 2 + >, 100 to 300 [mu] M of dNTPs, 0.3 to 0.8 M of betaine, 0.3 to 0.8 M of trehalose and 0.05 to 0.2 w / w% of Tween-20. According to the kit, a nucleic acid extraction step can be omitted, a blood sample is directly used for detection, the detection steps are simple and time-saving, and the kit has good sensitivity, specificity and kappa value.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to a G6PD deficiency screening kit and its application. Background Technology

[0002] G6PD deficiency (glucose-6-phosphate dehydrogenase deficiency) is a common inherited hemolytic disorder caused by mutations in the G6PD gene that lead to reduced or absent enzyme activity. Patients are prone to acute hemolysis upon exposure to certain medications, foods (such as fava beans), or infections, which can be life-threatening in severe cases. Currently, screening methods for G6PD deficiency mainly include enzyme activity testing and genetic diagnosis.

[0003] While enzyme activity assays are relatively simple to perform, they are easily affected by sample preservation conditions, testing environment, and individual physiological state, resulting in insufficient sensitivity and low specificity. False negatives or false positives are particularly common in newborns or special populations. Genetic diagnostics, with its high specificity and accuracy, has become an important supplementary method. However, its traditional procedures require nucleic acid extraction and purification, which are cumbersome, time-consuming, and dependent on specialized equipment and technicians, making it difficult to meet the needs of large-scale screening or rapid testing in primary healthcare institutions.

[0004] Furthermore, existing gene testing kits have limitations in sample compatibility. Most require purified nucleic acid samples, while direct testing of raw samples such as blood often results in decreased sensitivity and poor result stability due to interference from inhibitors (such as hemoglobin and proteins) in the amplification reaction. Therefore, developing a G6PD deficiency screening kit that requires no nucleic acid extraction, is easy to operate, and possesses both high sensitivity and specificity is of great significance for improving screening efficiency and expanding screening coverage.

[0005] The existing technology CN112442532B provides a primer set and kit for the simultaneous detection of 20 glucose-6-phosphate dehydrogenase deficiency diseases. It can simultaneously detect 20 pathogenic sites of glucose-6-phosphate dehydrogenase deficiency in a single reaction well. The 20 mutation types included in this invention can cover the pathogenic mutations carried by 99% of patients in the Chinese population. The detection is more comprehensive and can avoid missing double heterozygotes. It has the characteristics of high throughput, high accuracy, high sensitivity, fast detection speed and low detection cost. However, it still requires cell-free DNA as a template, that is, it requires an additional DNA extraction step, which is complicated to operate. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a G6PD deficiency screening kit and its application.

[0007] On one hand, the present invention provides a G6PD deficiency screening kit, the kit comprising a primer and probe set with sequences as shown in SEQ ID NO. 1-36.

[0008] Specifically, the probe has at least one of a fluorescent group and a quenching group.

[0009] More specifically, the fluorescent group includes, but is not limited to, at least one of the fluorescent groups FAM, ROX, VIC and CY5.

[0010] More specifically, the quenching groups include, but are not limited to, at least one of MGB, BHQ, and TAMRA.

[0011] Specifically, the G6PD deficiency screening kit also includes a fifth primer probe set for detecting an internal reference gene, which includes ACTB.

[0012] More specifically, the primer and probe sequence of the ACTB is shown in SEQ ID NO.37-39.

[0013] Specifically, the G6PD deficiency screening kit further includes a sample preservation solution, which comprises Mg 2+ dNTPs, betaine, trehalose and Tween-20.

[0014] More specifically, the sample preservation solution includes Mg 2+ 2.0-5.0 mM, dNTPs 100-300 μM, betaine 0.3-0.8 M, trehalose 0.3-0.8 M, and Tween-20 0.05-0.2 w / w.

[0015] Preferably, the sample preservation solution includes Mg 2+ 3.0-5.0 mM, dNTPs 200-300 μM, betaine 0.5-0.8 M, trehalose 0.3-0.5 M, and Tween-20 0.1-0.2 w / w.

[0016] Preferably, the sample preservation solution includes Mg 2+ 2.0-3.0 mM, dNTPs 200-300 μM, betaine 0.3-0.5 M, trehalose 0.5-0.8 M, and Tween-20 0.05-0.1 w / w.

[0017] Specifically, the steps for using the G6PD deficiency screening kit include: S1. Collect samples and preserve them using sample preservation solution; S2. Configure the amplification reaction system, use primer and probe sets for amplification, and detect whether the test sites in the sample have changed. S3. Determine whether G6PD deficiency exists based on the variation of the test site.

[0018] More specifically, in S1, the sample includes, but is not limited to, at least one of: serum, plasma, tissue, cultured cells, blood, feces, saliva, urine, or cell-free DNA.

[0019] More specifically, in S2, the amplification reaction system includes 8-10 parts of sample preservation solution containing the sample to be tested, 0.2-2 parts of primer mixture, and 4-5 parts of Taq, with ddH2O added to make up to 20 parts.

[0020] More specifically, in S2, the amplification program is as follows: 45-55℃, 2-4 min; 90-100℃, 0.5-1.5 min; 90-100℃, 1-3 s; 50-60℃, 10-20 s; 42 cycles.

[0021] In another aspect, the present invention provides the use of any component of the above-mentioned G6PD deficiency screening kit in the preparation of the G6PD deficiency screening kit.

[0022] Specifically, the components include a primer-probe set and a sample preservation solution.

[0023] Compared with the prior art, the present invention has the following advantages: The kit provided by this invention can directly test blood samples and achieve the same high sensitivity, specificity and kappa value as existing technologies for detecting cell-free DNA. Using the kit provided by this invention can significantly simplify the screening process and greatly reduce manpower and material costs. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0025] Example 1 The detection sites and corresponding primers in this embodiment are shown in Table 1: Table 1 Amplification Primer Sequences

[0026] Sample preservation solution: includes: Mg2+ 3.0 mM, dNTPs 200 μM, betaine 0.5 M, trehalose 0.5 M and Tween-20 0.1 w / w%, balance DEPC water.

[0027] Detection steps: Mix and dilute the blood sample with the sample preservation solution at a volume ratio of 1:10 to obtain the detection template. Use the primers in Table 1 for amplification. The amplification system is as follows: add 10 μL of detection template, primer mixture (the final concentration of each primer is 0.2 μM) and 5 μL of Novizan hot-start Taq enzyme P132 to a 20 μL reaction system, and make up the difference with ddH2O. Amplification and detection are performed. The amplification conditions are shown in Table 2.

[0028] Table 2

[0029] Blood samples were taken from 200 patients who signed informed consent forms and tested according to the above steps, with Sanger sequencing results used as a control. The test results are shown in Table 3. Table 3

[0030] Sensitivity is 100%, specificity is 100%, and Kappa value is 100%.

[0031] Example 2 The sample preservation solution, consistent with the primers used in Example 1, comprises: Mg 2+ 5.0 mM, dNTPs 300 μM, betaine 0.8 M, trehalose 0.3 M and Tween-20 0.2 w / w%, balance DEPC water.

[0032] Detection steps: Mix and dilute the blood sample with the sample preservation solution at a volume ratio of 1:5 to obtain the detection template. Use the primers in Table 2 for amplification. The amplification system is as follows: add 10 μL of detection template, primer mixture (the final concentration of each primer is 0.2 μM) and 5 μL of Novizan hot-start Taq enzyme P132 to a 20 μL reaction system, and make up the difference with ddH2O. Perform amplification and detection. The amplification conditions are shown in Table 2.

[0033] The blood sample obtained in Example 1 was tested according to the above steps, and Sanger sequencing was performed as a control. The test results are shown in Table 4: Table 4

[0034] Sensitivity is 100%, specificity is 100%, and Kappa value is 100%.

[0035] Example 3 The sample preservation solution, consistent with the primers used in Example 1, comprises: Mg 2+ 2.0 mM, dNTPs 100 μM, betaine 0.3 M, trehalose 0.8 M and Tween-20 0.05 w / w%, balance DEPC water.

[0036] Detection steps: Mix and dilute the blood sample with the sample preservation solution at a volume ratio of 1:20 to obtain the detection template. Use the primers in Table 2 for amplification. The amplification system is as follows: add 10 μL of detection template, primer mixture (the final concentration of each primer is 0.2 μM) and 5 μL of Novizan hot-start Taq enzyme P132 to a 20 μL reaction system, and make up the difference with ddH2O. Perform amplification and detection. The amplification conditions are shown in Table 2.

[0037] The blood sample obtained in Example 1 was tested according to the above steps, and Sanger sequencing was performed as a control. The test results are shown in Table 5: Table 5

[0038] Sensitivity is 100%, specificity is 100%, and Kappa value is 100%.

[0039] Comparative Example 1 The difference from Example 1 is that the sample preservation solution includes: Mg 2+ 3.0 mM, dNTPs 200 μM, trehalose 1 M and Tween-20 0.1 w / w%, balance DEPC water.

[0040] Detection steps: Mix and dilute the blood sample with the sample preservation solution at a volume ratio of 1:5 to obtain the detection template. Use the primers in Table 2 for amplification. The amplification system is as follows: add 10 μL of detection template, primer mixture (the final concentration of each primer is 0.2 μM) and 5 μL of Novizan hot-start Taq enzyme P132 to a 20 μL reaction system, and make up the difference with ddH2O. Perform amplification and detection. The amplification conditions are shown in Table 2.

[0041] The blood sample obtained in Example 1 was tested according to the above steps, and Sanger sequencing was performed as a control. The test results are shown in Table 6: Table 6

[0042] The sensitivity is 80%, the specificity is 85%, and the Kappa value is 65%.

[0043] Comparative Example 2 The difference from Example 1 is that the sample preservation solution includes: Mg 2+3.0 mM, dNTPs 200 μM, betaine 1 M and Tween-20 0.1 w / w%, balance DEPC water.

[0044] Detection steps: Mix and dilute the blood sample with the sample preservation solution at a volume ratio of 1:5 to obtain the detection template. Use the primers in Table 2 for amplification. The amplification system is as follows: add 10 μL of detection template, primer mixture (the final concentration of each primer is 0.2 μM) and 5 μL of Novizan hot-start Taq enzyme P132 to a 20 μL reaction system, and make up the difference with ddH2O. Perform amplification and detection. The amplification conditions are shown in Table 2.

[0045] The blood sample obtained in Example 1 was tested according to the above steps, and Sanger sequencing was performed as a control. The test results are shown in Table 7: Table 7

[0046] The sensitivity is 75%, the specificity is 80%, and the Kappa value is 55%.

[0047] Comparative Example 3 The difference from Example 1 is that the sample preservation solution includes Mg 2+ 3.0 mM, dNTPs 200 μM and Tween-20 0.1 w / w%, balance DEPC water.

[0048] Detection steps: Mix and dilute the blood sample with the sample preservation solution at a volume ratio of 1:5 to obtain the detection template. Use the primers in Table 2 for amplification. The amplification system is as follows: add 10 μL of detection template, primer mixture (the final concentration of each primer is 0.2 μM) and 5 μL of Novizan hot-start Taq enzyme P132 to a 20 μL reaction system, and make up the difference with ddH2O. Perform amplification and detection. The amplification conditions are shown in Table 2.

[0049] The blood sample obtained in Example 1 was tested according to the above steps, and Sanger sequencing was performed as a control. The test results are shown in Table 8: Table 8

[0050] The sensitivity is 60%, the specificity is 70%, and the Kappa value is 30%.

[0051] Comparative Example 4 The difference from Example 1 is that the sample preservation solution includes: Mg 2+ 3.0 mM, dNTPs 200 μM, betaine 0.5M, sucrose 0.5M and Tween-20 0.1 w / w%, balance DEPC water.

[0052] Detection steps: Mix and dilute the blood sample with the sample preservation solution at a volume ratio of 1:5 to obtain the detection template. Use the primers in Table 2 for amplification. The amplification system is as follows: add 10 μL of detection template, primer mixture (the final concentration of each primer is 0.2 μM) and 5 μL of Novizan hot-start Taq enzyme P132 to a 20 μL reaction system, and make up the difference with ddH2O. Perform amplification and detection. The amplification conditions are shown in Table 2.

[0053] The blood sample obtained in Example 1 was tested according to the above steps, and Sanger sequencing was performed as a control. The test results are shown in Table 9: Table 9

[0054] The sensitivity was 78%, the specificity was 83%, and the Kappa value was 61%.

[0055] Detection Example 1 Plasmids carrying variant sites were constructed, diluted with blood samples, and detected according to the methods of Examples 1-3 or Comparative Examples 1-4. The sensitivity test results are shown in Tables 10-11.

[0056] Table 10

[0057] Table 11

[0058] The above data demonstrate that using the kit provided by this invention can effectively improve the sensitivity of detection.

[0059] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A G6PD deficiency screening kit, characterized in that, The kit includes a set of primers and probes with sequences as shown in SEQ ID NO.1-36.

2. The G6PD deficiency screening kit according to claim 1, characterized in that, The probe contains at least one of a fluorescent group and a quenching group.

3. The G6PD deficiency screening kit according to claim 2, characterized in that, The fluorescent group includes at least one of FAM, VIC and CY5 fluorescent groups.

4. The G6PD deficiency screening kit according to claim 2, characterized in that, The quenching group includes at least one of MGB, BHQ and TAMRA.

5. The G6PD deficiency screening kit according to claim 1, characterized in that, It also includes a fifth set of primers and probes for detecting an internal reference gene, which includes ACTB.

6. The G6PD deficiency screening kit according to claim 1, characterized in that, It also includes a sample preservation solution, which comprises Mg 2+ dNTPs, betaine, trehalose and Tween-20.

7. The G6PD deficiency screening kit according to claim 6, characterized in that, The sample preservation solution includes Mg 2+ 2.0-5.0 mM, dNTPs 100-300 μM, betaine 0.3-0.8 M, trehalose 0.3-0.8 M, and Tween-20 0.05-0.2 w / w.

8. The G6PD deficiency screening kit according to claim 7, characterized in that, The sample preservation solution includes Mg 2+ 3.0-5.0 mM, dNTPs 200-300 μM, betaine 0.5-0.8 M, trehalose 0.3-0.5 M, and Tween-20 0.1-0.2 w / w.

9. The G6PD deficiency screening kit according to claim 7, characterized in that, The sample preservation solution includes Mg 2+ 2.0-3.0 mM, dNTPs 200-300 μM, betaine 0.3-0.5 M, trehalose 0.5-0.8 M, and Tween-20 0.05-0.1 w / w.

10. The G6PD deficiency screening kit according to any one of claims 7-9, characterized in that, The usage steps include: S1. Collect samples and preserve them using sample preservation solution; S2. Configure the amplification reaction system, use primer and probe sets for amplification, and detect whether the test sites in the sample have changed. S3. Determine whether G6PD deficiency exists based on the variation of the test site.

11. The G6PD deficiency screening kit according to claim 10, characterized in that, In S1, the sample includes at least one of serum, plasma, tissue, cultured cells, blood, feces, saliva, urine, or cell-free DNA.

12. The G6PD deficiency screening kit according to claim 10, characterized in that, In S2, the amplification reaction system includes 8-10 parts of sample preservation solution containing the sample to be tested, 0.2-2 parts of primer mixture, and 4-5 parts of Taq, with ddH2O added to make up to 20 parts.

13. The G6PD deficiency screening kit according to claim 10, characterized in that, In S2, the amplification program is as follows: 45-55℃, 2-4 min; 90-100℃, 0.5-1.5 min; 90-100℃, 1-3 s; 50-60℃, 10-20 s; 42 cycles.

14. The use of any component of the G6PD deficiency screening kit according to any one of claims 1-13 in the preparation of the G6PD deficiency screening kit.

15. The application according to claim 14, characterized in that, The components include a primer and probe set and a sample preservation solution.

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