CN-121992122-A - Wheatstone's-shaped body high-specificity detection primer group based on whole genome precise screening and application thereof

Abstract

The invention belongs to the technical field of biological detection, and relates to a Whipple barrier high-specificity detection primer set based on whole genome precise screening and application thereof. Aiming at the problem that a common target in the prior art is easy to generate nonspecific amplification under a complex background, the invention eliminates human and environmental microorganism interference sequences from the source by integrating a very large-scale microorganism database for accurate calculation, and screens to obtain a specific primer pair SEQ ID NO.2-3. The primer group has excellent specificity, does not need probe auxiliary specificity, and can realize extremely high signal-to-noise ratio and sensitivity (100 copies/mL) only in a dye-process qPCR system. The invention solves the false positive pain point in complex clinical sample detection, is not only suitable for conventional fluorescent quantitative PCR, but also provides core target support for detection technologies such as digital PCR and the like, and provides reliable technical guarantee for clinical accurate detection and early diagnosis of the Huipple barrier.

Inventors

• LIU CHANG
• FU XU
• MA MIAO
• SHI JIPING

Assignees

• 南京鼓楼医院

Dates

Publication Date

20260508

Application Date

20260127

Claims (10)

1. 1. A Wheatstone's-support high-specificity detection primer set based on whole genome precise screening is characterized by comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 3.
2. 2. A huipple's barrier high specificity detection kit based on whole genome computational screening, characterized in that the kit comprises the primer set of claim 1.
3. 3. The kit of claim 2, further comprising PCR MASTER Mix, wherein said PCR MASTER Mix comprises SYBR Green I, taq enzyme.
4. 4. The kit of claim 2, wherein the concentration of each primer in the kit is independently 10 μm.
5. 5. The kit of claim 2, further comprising a negative control and a positive control; the negative control is TE buffer; The positive reference substance is a recombinant plasmid containing a target gene sequence of a Wheatstone nutrient, and the vector skeleton is PUC18.
6. 6. A method of detecting a whipple support, comprising the steps of: 1) Extracting DNA of a sample to be detected; 2) Performing fluorescent quantitative PCR amplification reaction on the DNA of the sample to be detected extracted in the step 1) by using the kit according to any one of claims 2 to 5; 3) Detecting the amplification reaction product in the step 2), and carrying out qualitative judgment and/or quantitative detection on the condition that the sample to be detected contains the Whipple-type barrier according to the detection result.
7. 7. The method according to claim 6, wherein in step 1), a DNA extraction kit is used to extract DNA from the sample to be tested; The sample to be detected comprises sputum, alveolar lavage fluid, cerebrospinal fluid or oropharyngeal swab.
8. 8. The method according to claim 6, wherein in the step 2), the reaction system for fluorescent quantitative PCR amplification comprises the following components in a total volume of 20. Mu.L, 2 Xdye method PCR MASTER Mix 10. Mu.L, 10. Mu.M forward primer 0.8. Mu.L, 10. Mu.M reverse primer 0.8. Mu.L, DNA template 1.0. Mu.L, ddH 2 O7.4. Mu.L.
9. 9. The method according to claim 6, wherein in step 3), the amplification reaction product is detected and a qualitative determination is made based on the detection result by a method selected from the group consisting of gel electrophoresis, fluorescent dye observation and amplification curve analysis.
10. 10. Use of a primer set according to claim 1, or a kit according to one of claims 2 to 5, or a method according to one of claims 6 to 9, for the preparation of a reagent for the detection of a hewlett-packard organism.

Description

Wheatstone's-shaped body high-specificity detection primer group based on whole genome precise screening and application thereof Technical Field The invention belongs to the technical field of biological detection, and particularly relates to a Whipple barrier high-specificity detection primer set, a kit and a method based on whole genome precise screening. Background The Huipol organism (Tropheryma whipplei, TW) is a gram-positive actinomycete, widely existing in natural environments such as soil and sewage, and is a pathogen causing rare systemic infectious diseases, namely Whipple's Disease (WD). The strain has strong invasiveness, and although the in-vitro culture difficulty is extremely high, the strain can be spread to the whole body through the field implantation of human epithelium and endothelial cells, and the infection is not limited to a single system, but is characterized by multiple organ involvement as a typical characteristic, thus the strain has a significant challenge to clinical diagnosis. From the clinical phenotype, the first and core affected parts of TW infection are often digestive systems, intestinal lesions are the most representative manifestations of Whipple disease, and bacteria are initially planted in small intestinal mucosa to cause intestinal lipodystrophy, so that chronic diarrhea (most of diarrhea), abdominal pain, belly lymphadenectasis and other symptoms are caused, and problems such as weight collapse, malnutrition, anemia and the like can be caused for a long time, and even complications such as hepatosplenomegaly, ascites and the like occur in severe cases. Notably, digestive symptoms often appear in late stages of the disease, while early stages are more common in which skeletal muscle systems are involved—patients may develop wandering joint pain or arthritis, often involve a single large joint, and are highly susceptible to missed or misdiagnosis due to similar symptoms to rheumatoid arthritis and negative detection of rheumatoid factors. Along with the progress of the disease, TW can be spread to key organs such as central nervous system, circulatory system and respiratory system through a lymphatic system, wherein serious symptoms such as cognitive dysfunction, mental abnormality, eye muscle paralysis and the like can be caused by the involvement of the nervous system, dementia-eye muscle paralysis-myoclonus triple sign is the characteristic expression of the disease, the circulatory system can cause blood culture negative infectious endocarditis, the progress is hidden but life is endangered, symptoms such as pneumonia, chronic cough, pleural adhesion and the like can be caused by the respiratory system which is not a main target organ, and severe pneumonia can even directly threaten the life of a patient. In addition, skin mucosa pigmentation, uveitis, epididymitis and other rare symptoms are also rarely reported, and the characteristic of 'multisystem invasion' is further highlighted. For the clinical detection of TW, the existing methods have obvious limitations, which also make the timely diagnosis of the disease particularly difficult. Traditional tissue staining methods (such as PAS staining and Grocott hexamine silver staining) are the first choice for diagnosis, but have low sensitivity and poor specificity, and low bacterial specimens or sparse positive cell distribution are easy to generate false negative, and pathogens such as mycobacterium tuberculosis and the like can cause false positive, and even if the result is positive, other methods are needed to verify. Although Immunohistochemistry (IHC) specificity is superior to staining, early detection and retrospective analysis can be achieved, but the risk of leakage-proof diagnosis cannot be fully specified. The advent of molecular biology methods provides a new path for TW detection, wherein mNGS can rapidly detect pathogenic bacteria and can obtain results within one day, but the detection cost is high and large-scale clinical application is not possible. In contrast, the real-time fluorescence quantitative PCR (qPCR) technology is mature, the detection cost is low, the operation is convenient, and the TW detection method is more suitable for clinical popularization. However, the accuracy of the technology is highly dependent on primer design, and the property of TW brings natural obstacle to primer development, namely, because the strain is difficult to culture in vitro, the early available whole genome sequencing data is extremely small, so that the designed primer lacks population representativeness, amplification failure or false negative easily occurs due to strain difference, and the application value of qPCR in clinic is severely limited. Fluorescent quantitative PCR is largely classified into two types, namely, a dye method without a probe and a probe method with a probe. The core requirement of the qualified primer is that the primer has high specificity and no non-specific amplification sh