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Primer, probe, kit and method for detecting HLA deletion type recurrence of patient

A deletion-type and kit-based technology, applied in the field of genetic engineering, can solve the problem of inability to benefit from lymphocyte infusion, and achieve the effects of easy interpretation of results, simple operation, and high type coverage

Inactive Publication Date: 2020-12-25
SHENZHEN TISSUEBANK PRECISION MEDICINE CO LTD +3
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows quicker and more precise identification of individuals who are at risk or have relapse after being treated by an immune therapy treatment called Lyme disease (LB). It offers various technical benefits such as good type coverage, stability during use, ease of handling, and interpretability on data obtained through analysis techniques like PCR amplification testing.

Problems solved by technology

This patented technical problem addressed by this patents relates to finding better ways to detect if someone who may have recovered their own blood system due to another person's disease called hemolytic crisis syndrome. Hemolysis disorders are caused when certain parts of red blood cells break down too much sugar into water instead of producing energy needed for normal body functions like oxygen production during metabolisms. However, these conditions cannot happen naturally because they occur frequently enough at once without causing damage to healthy tissues such as bone marrow nerves. Current diagnostic techniques involve testing multiple samples before making conclusion on whether any given sample contains more than usual levels of specific gene sequences associated with bleeding complications.

Method used

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  • Primer, probe, kit and method for detecting HLA deletion type recurrence of patient
  • Primer, probe, kit and method for detecting HLA deletion type recurrence of patient
  • Primer, probe, kit and method for detecting HLA deletion type recurrence of patient

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Example 1: Specificity of Primer Pairs and Probes for HLA Deletion Recurrence Detection

[0058] In this embodiment, primer pairs and probes are designed for specific HLA types, and the specificity of the primer pairs and probes is judged by the Ct value of the fluorescent quantitative PCR reaction.

[0059] In the present invention, samples with a Ct value of ≤30 are judged as positive, and samples with a Ct value of ≥40 or no amplification curve are judged as negative.

[0060] combined with figure 1 , from the amplification curves of positive and negative samples of specific HLA types A*02, B*13, C*01 and C*07, it can be seen that the Ct values ​​of positive samples are 25.2, 26.9, 29.6 and 28.1 respectively, Negative specimens show no amplification. It shows that the above-mentioned types of primer pairs and probes have good specificity.

Embodiment 2

[0061] Example 2: Amplification Efficiency of HLA Deletion Recurrence Detection Primer Pairs and Probes

[0062] In this example, laboratory donor samples of HLA-A*02, B*13, C*01, and C*07 types detected by next-generation sequencing were selected as positive templates, and samples without this type were regarded as negative.

[0063] The positive and negative DNA samples were diluted to 15ng / μl, and the negative sample DNA was used to perform a 5-fold serial dilution of the positive sample. The positive samples (100%, 20%, 4%, 0.8%, 0.16%) were used for qPCR detection, and each sample was replicated three times, and the standard curve was drawn using the positive sample concentration and the average value of Ct.

[0064] combined with figure 2 , it can be seen that the above site standard curve correlation coefficient R 2 >0.99, confirming that the primers amplified linearly within the range of 0.16%-100%.

[0065] by attaching figure 2 The slope of the amplification cu...

Embodiment 3

[0066] Example 3: Sensitivity of HLA Deletion Recurrence Detection Primer Pairs and Probes

[0067] In this example, HLA-A*01, A*02, A*11, A*23, A*25, B*13, B*35, B*38, C*01, C*02 detected by first-generation sequencing were selected. , C*03, C*04, C*05, C*07, DRB1*04, DRB1*12, DQB1*02, DQB1*04, DQB1*05, DPB1*01:01, DPB1*02:01, DPB1 *04:01 and DPB1*05:01 laboratory donor samples were taken as positive templates, and samples without this type were taken as negative.

[0068] The DNA concentrations of the above samples were all diluted to 15ng / μl, and the positive samples were diluted to 0.16% with the negative samples, and 2 batches were tested every day for 5 consecutive days.

[0069] The detection results are shown in Table 5. The coefficients of variation of the above-mentioned type primer pairs and probes between batches and within batches of Ct values ​​are all less than 5%, and the detection results of A*02, B*13, C*01 and C*07 are as follows: attached image 3 As sho...

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Abstract

The invention belongs to the technical field of gene engineering, and discloses a primer and probe combination for detecting HLA deletion type recurrence of a patient, a kit containing the primer andprobe combination, and a method for detecting HLA deletion type recurrence of the patient by adopting the primer and probe combination or the kit. The primer and probe combination is designed for specific HLA types on the basis of the latest IMGT database, the types of transplantation recurrence patients can be accurately identified, and a clinical treatment scheme is guided. Compared with the prior art, the method disclosed by the invention has the advantages of high coverage, stable performance, high sensitivity, simplicity in operation, easiness in result interpretation and the like.

Description

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Claims

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Application Information

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Owner SHENZHEN TISSUEBANK PRECISION MEDICINE CO LTD
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