Application of a set of cfDNA sequences as a risk assessment marker for infection with rhizomucor malenophilia

Abstract

The application relates to the technical field of serological detection evaluation, and particularly discloses application of a group of cfDNA sequences as markers for evaluating the risk of infection of Coccidioides immitis, a serological detection method thereof has the advantages of high sensitivity, high specificity, non-invasiveness and short time consumption, and the detection result can be used as intermediate data and is suitable for evaluating reference of invasive pulmonary aspergillosis. The marker sequence is short (100-200 bp), high in abundance, high in conservation and low in fungal homology, and is widely present in blood circulation, so that sampling is non-invasive. The detection is carried out through qPCR combined with specific primer probes (SEQ ID NO. 4-12, the probe contains a fluorescent reporter / quenching group), and the performance verification shows that the sensitivity is high, the specificity and the AUC value are better than those of a traditional ELISA method, the blood mcfDNA detection can be realized, and the whole-process risk evaluation of infection is supported.

Description

Technical Field

[0001] This invention relates to the field of serological testing, and more specifically, to the application of a set of cfDNA sequences as biomarkers for assessing the risk of *Basilella marneffei* infection. Background Technology

[0002] Invasive fungal infections are a growing public health challenge worldwide, among which *Basilella marneffei* (…) Talaromyces marneffei Penicillium marneffei (formerly known as Penicillium marneffei) is an important dimorphic opportunistic pathogenic fungus prevalent in Southeast Asia and can spread widely with population movement. This bacterium is particularly pathogenic to immunocompromised individuals (such as HIV-infected individuals and organ transplant recipients), potentially leading to disseminated and fatal infections.

[0003] Current technologies face significant challenges in auxiliary research and risk assessment of *Brachysmus marneffei* infection. On one hand, *Brachysmus marneffei* can colonize the human respiratory tract, overlapping clinical manifestations with invasive infections. On the other hand, traditional detection methods have significant shortcomings. For example, fungal culture is time-consuming and has low sensitivity; histopathological examination is invasive, and molecular testing based on respiratory samples (such as bronchoalveolar lavage fluid, BALF) is also highly invasive and carries the risk of false negatives due to inaccurate localization, and cannot effectively distinguish between colonization and active infection. While blood sample detection technologies can circumvent these shortcomings, current methods suffer from limited sample sizes, stringent experimental conditions, and a lack of specific detection methods for small DNA fragments among publicly available methods for detecting exogenous nucleic acid components in blood. This limits their application in research or preclinical screening and hinders in-depth research into the infection mechanisms of *Brachysmus marneffei*.

[0004] In recent years, circulating free DNA (cfDNA), especially exogenous microbial circulating free DNA (mcfDNA), has become a cutting-edge biomarker for infectious disease risk assessment. mcfDNA originates directly from nucleic acid fragments released by the immune system during pathogen cleavage during infection. Its detection in peripheral blood specifically points to active infection rather than colonization, and its load is closely related to the severity of infection. Although this technology has shown great potential in bacterial, viral, and even some fungal infections, its application in the risk assessment of Marneffei basidium infection remains largely unexplored. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of at least one of the above-mentioned prior art and provide a set of cfDNA sequences as biomarkers for assessing the risk of *Basilella marneffei* infection. This biomarker combination can be used to prepare a detection kit, and its serological detection has the advantages of high sensitivity, high specificity, and non-invasiveness. The detection results can be used as intermediate data for reference.

[0006] The technical solution adopted in this invention is to provide a reagent for quantitatively detecting the biomarker cfDNA in the preparation of a *Brachysmus marneffei* infection risk assessment product, wherein the sequence of the biomarker cfDNA includes at least one of SEQ ID NO. 1 to 3. SEQ ID NO. 1 to 3 are respectively selected from the conserved genes TMATCC_006500, TMATCC_000757, and TMATCC_001136 of *Brachysmus marneffei*.

[0007] Further, the reagents for quantitative detection of biomarkers tRFs include at least one of the following primers and probes: TMATCC_006500_F, TMATCC_006500_R, TMATCC_006500_P, TMATCC_000757_F, TMATCC_000757_R, TMATCC_000757_P, TMATCC_001136_F, TMATCC_001136_R, and TMATCC_001136_P, the sequences of which are shown in SEQ ID NO.4~12.

[0008] Furthermore, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group.

[0009] Further, the fluorescent reporter group includes at least one of FAM, VIC, HEX, TET, JOE, CY3 and CY5; and / or, the fluorescent quencher group includes at least one of TAMARA, MGB, BHQ-1, BHQ-2 and BHQ-3.

[0010] Furthermore, the risk assessment includes the following steps:

[0011] S1: Collect plasma samples from patients and extract total cfDNA; S2: Using the biomarker cfDNA as a target, the total cfDNA is detected by qPCR to obtain the CT value; S3: Based on the comparison between the measured CT value and the preset threshold, the risk of the patient being infected with *Brachysmus marneffei* is determined.

[0012] Furthermore, the preset threshold mentioned in step S3 is defined as follows: C1: If the CT value of the biomarker is ≤37.5 in qPCR detection, the risk of *Basilella marneffei* infection is relatively high; C2: If the CT value of the biomarker is >37.5 in qPCR detection, the risk of *Basilella marneffei* infection is low.

[0013] Furthermore, the *Basilaria marneffei* infection includes disseminated *Basilaria marneffei* infection.

[0014] Current PCR protocols for detecting *Cladosporium marneffei* in humans have a major drawback: the nucleic acid sequence of the sample must be at least 500 bp. Therefore, existing techniques require tissue or BALF (bronchial lavage fluid) samples, which carries drawbacks including invasiveness, false negatives, and inability to distinguish infected areas. While blood sample detection techniques can circumvent these drawbacks, current methods suffer from limited sample sizes, stringent experimental conditions, and a lack of specific detection methods for small DNA fragments among publicly available methods for detecting exogenous nucleic acid components in blood, making it difficult to detect *mcfDNA* in blood.

[0015] Based on the above-mentioned problems, in one or more embodiments of the present invention, the inventors used sequencing technology to screen and obtain three cfDNA nucleic acid fragments from the conserved genes TMatcc_006500, TMatcc_001136, and TMatcc_000757 of *Brachys malneffei*. These fragments have the advantages of high abundance, high conservation, and low fungal homology, with sequence lengths of approximately 100-200 bp. They are short nucleic acid sequence fragments widely present in the blood circulation of patients, and sampling is relatively non-invasive. Then, amplification primers and signal reporter probes were designed using these sequences as target sequences, with nucleotide sequences shown in SEQ ID NO. 4-12. The risk assessment efficacy of the above primer and probe combination was verified in the embodiments, demonstrating high detection sensitivity, specificity, and AUC values ​​higher than those of the traditional ELISA method. Furthermore, since cfDNA is an exogenous nucleic acid fragment in cell lysates produced by the body after the host infection with *Brachys malneffei* and the resulting immune response, this primer and probe combination can be used for the full-process monitoring of *Brachys malneffei* infection, especially for early infection risk assessment.

[0016] Another object of the present invention is to provide a kit for detecting *Basilella marneffei*, comprising at least one of the following primers and probes: TMATCC_006500_F, TMATCC_006500_R, TMATCC_006500_P, TMATCC_000757_F, TMATCC_000757_R, TMATCC_000757_P, TMATCC_001136_F, TMATCC_001136_R, and TMATCC_001136_P, wherein the sequences of the primers and probes are shown in SEQ ID NO. 4~12.

[0017] Furthermore, the kit also includes sample nucleic acid extraction reagents and host-derived nucleic acid elution reagents.

[0018] Another object of the present invention is to provide a detection system for assessing the risk of *Cladosporium marneffei* infection, the detection system comprising: The detection device is used to detect the level of the biomarker cfDNA in patient biological samples; The comparison device compares the measured level of the biomarker cfDNA with a preset threshold to determine whether the patient has a Basiliformis marneffei infection. The biomarker cfDNA is derived from genes TMatcc_006500, TMatcc_001136 and / or TMatcc_000757, and its sequence is shown in SEQ ID NO.1~3.

[0019] Furthermore, the preset threshold is defined as follows: C1: If the CT value of the biomarker is ≤37.5 in qPCR detection, the risk of *Basilella marneffei* infection is relatively high; C2: If the CT value of the biomarker is >37.5 in qPCR detection, the risk of *Basilella marneffei* infection is low.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a set of cfDNA sequences as biomarkers for assessing the risk of *Basilella marneffei* infection. The serological detection method offers advantages such as high sensitivity, high specificity, non-invasiveness, and short processing time. The detection results can serve as intermediate data and are suitable for assessing invasive pulmonary aspergillosis. These biomarker sequences are short (100-200 bp), highly abundant, highly conserved, and have low fungal homology. They are widely present in the bloodstream, and sampling is non-invasive. Detection using qPCR combined with specific primers and probes (SEQ ID NO. 4-12, probes containing fluorescent reporter / quencher groups) demonstrates high sensitivity, specificity, and AUC values ​​superior to traditional ELISA methods, enabling the detection of mcfDNA in blood and supporting risk assessment throughout the infection process. Attached Figure Description

[0021] Figure 1 This is a frequency distribution diagram of *Bacteroides marneffei* cfDNA genes. Among them, the genes TMATCC_006500, TMATCC_001136, and TMATCC_000757 rank 1st, 2nd, and 7th in frequency, respectively.

[0022] Figure 2 This is a multicomponent qPCR fluorescence detection result plot. The plot uses cycle number as the horizontal axis and fluorescence intensity as the vertical axis, showing the dynamic changes in fluorescence signals of the detected components during real-time fluorescence detection. In the legend, the blue curve represents the FAM-labeled components from eight suspected *Brachysmus marneffei* infection patients, and the red curve represents the ROX-labeled passive reference. The dynamic curves show that the four cases with increased fluorescence intensity can be assessed as having a high risk of *Brachysmus marneffei* infection.

[0023] Figure 3 The relationship between the fluorescence signal change value ΔRn and the cycle number during qPCR for different samples is shown. Among them, the ΔRn of 4 curves shows logarithmic growth, indicating that the DNA of the tested object has entered the exponential amplification stage. This corresponds to 4 of the 8 suspected patients who can be assessed as having a high risk of Bacteroides marneffei infection.

[0024] Figure 4 The ROC curves represent the risk assessment efficacy of the hybrid multiplex qPCR assay and the ELISA assay. The blue line represents the hybrid multiplex qPCR assay for cfDNA, with AUC=0.738, sensitivity 0.70, and specificity 0.70; the red line represents the ELISA assay, with AUC=0.677, sensitivity 0.733, and specificity 0.50. Detailed Implementation

[0025] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] Sources of reagents, materials, and equipment: Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all experimental methods are conventional experimental methods in this field.

[0028] 1. Establish an in vitro immune cell model of *Basilella marneffei* infection. Materials: human immune cell mixture, *Basilella marneffei* strain AF293 (ATCC), PRMI 1640 medium (GIBCO), and premium Australian fetal bovine serum (GIBCO).

[0029] Experimental method: Immune cells were extracted from whole blood of healthy individuals, with a total cell concentration reaching 3... 10 7 / mL, add Bacteroides marneffei ATCC18224 cells, at 3 10 6 Samples of the supernatant were taken after culturing high concentrations of *Bacteroides marneffei* ( / mL) for 2, 4, 6, 8, and 10 hours.

[0030] 2. Sample collection and purification Materials and instruments: cfDNA preservation solution, MagicPure® Cell-Free DNA Kit II magnetic bead extraction kit for cfDNA, Aamp DNA Microbiome Kit host-derived nucleic acid elution kit.

[0031] Experimental methods: cfDNA was extracted from the supernatant of the co-culture product using a magnetic bead extraction kit. The cfDNA product was purified using a cfDNA purification kit, and host-derived nucleic acid fragments were eluted from the cfDNA using a host-derived nucleic acid elution kit. The purified cfDNA was stored in cfDNA preservation solution at 2-8℃ and next-generation sequencing was performed within 3 days at 2-8℃ (Guangzhou Micro-Gene Co., Ltd.).

[0032] 3. Sequencing of cfDNA Materials and instruments: NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, IlluminaNextSeq 2000 sequencer.

[0033] Experimental Methods: DNA libraries were constructed using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, which involved DNA fragmentation, end repair, adapter ligation, and PCR amplification. Quality control measures, including DNA concentration assays, agarose gel electrophoresis, and fragment length determination, were performed to complete library construction. DNA nanospheres (DNBs) were prepared and sequenced using the Illumina platform.

[0034] 4. Sequencing data analysis (1) The raw reads obtained from sequencing need to be strictly quality controlled. High-quality clean reads are obtained by removing read pairs containing adapter sequences, fragments with N content exceeding 10% in single-end reads, or low-quality bases (Q≤5) accounting for more than 50%.

[0035] (2) The genome is indexed and compared with the nucleic acid sequence of the whole species. The corresponding gene position of the corresponding cfDNA fragment in *Basilus marneffei* is obtained by blast. Repetitive and fragmented nucleic acid fragments are removed. Finally, the top 500 cfDNA fragments with the highest expression levels and their corresponding genome positions are selected and the results are output.

[0036] 5. PCR primer design Based on the genes obtained after sequencing and screening, primer capture designs were conducted using the highly expressed cfDNA fragments. The specific design principles are as follows: Primer lengths are designed to be between 15 and 30 bases; The primers have a GC content between 40% and 60% and a Tm value close to 72℃. The 3′ end of the primer avoids the third position of the codon, and T is preferred at the 3′ end of the primer, with bases randomly distributed; There should be no complementary sequences between primers themselves or between primers; The ΔG values ​​at different positions can be analyzed using Oligo6 software. The ΔG values ​​at the 5′ end and the middle of the primer should be higher than those at the 3′ end. The single strands of the amplified product cannot form secondary structures.

[0037] 6. Research subjects All participants in this study were recruited from inpatients and outpatients of the Department of Respiratory Medicine at the First Affiliated Hospital of Guangzhou Medical University. The study period was from January 2020 to December 2025.

[0038] All subjects in the cohort completed etiological evidence testing (the gold standard for diagnosis), with no sample dropouts or missing data. All test results were included in subsequent statistical analysis. Etiological evidence testing methods included: isolating and identifying pathogens from specimens such as patient blood, bone marrow, lymph nodes, or skin biopsies through fungal culture; or identifying pathogens within or outside macrophages through histopathology or direct microscopic examination.

[0039] In the initial validation cohort, eight patients suspected of having *Basilella marneffei* infection were included to preliminarily validate the ability of the pooled multiplex qPCR assay to detect *Basilella marneffei* cfDNA. Following pooled multiplex qPCR testing and etiological evidence analysis, four cases were confirmed to be *Basilella marneffei* infection, while the other four were infected with other fungi (Aspergillus or Cryptococcus).

[0040] In the risk assessment efficacy validation cohort, 60 patients suspected of having *Gnaphalium marneffei* infection were also included to compare the risk assessment efficacy of the mixed multiplex qPCR and ELISA methods. Following mixed multiplex qPCR and ELISA testing, and further pathogen evidence testing, 33 cases were confirmed to have *Gnaphalium marneffei* infection, meeting the criteria of the *Guidelines for the Assessment and Treatment of Invasive Pulmonary Fungal Diseases (2025 Edition)*; the remaining 27 cases were infected with other fungi (Aspergillus or Cryptococcus) and / or specific pathogens. There were no statistically significant differences in baseline characteristics such as age, sex, and underlying diseases between the two groups (P>0.05), making them comparable.

[0041] 7. qPCR Materials: qPCR amplification reaction system, magnetic bead-based cfDNA extraction and purification kit, and plasma samples from 8 patients with Marneffei basket disease. The amplification system and conditions are shown in the table below.

[0042] Table 1. PCR amplification reaction system

[0043] 9. ELISA Materials and instruments: Microplate reader; Penicillium marneffei antibody (PM Ab) ELISA kit, including microplate, pipette, washing buffer, blocking buffer, antigen protein, antibody protein, luminescent substrate, stop solution, etc.

[0044] Methods: The Mplp protein, a specific polysaccharide antigen of the cell wall of Penicillium marneffei, was detected using a Penicillium marneffei antibody (PM Ab) ELISA kit.

[0045] 10. Statistical Analysis 1) Baseline data comparison: The baseline data such as age and gender of the two groups of subjects were compared. For normally distributed continuous data, the independent samples t test was used, for non-normally distributed data, the Man-Whitney U test was used, and for count data, the χ² test was used. P>0.05 was considered as no statistical difference.

[0046] 2) Risk assessment efficacy analysis: The assessment value of each indicator was analyzed using receiver operating characteristic (ROC) curves. The area under the curve (AUC) and 95% confidence interval (95% CI) were calculated. The optimal assessment cutoff value, i.e., the CT value of qPCR, was determined based on the maximum value of the Youden index. At the same time, the corresponding sensitivity, specificity, and accuracy were calculated.

[0047] 3) Statistical software and significance criteria: All statistical analyses were performed using GraphPad Prism 9.0 and R language 4.2.1 software to plot ROC curves; P < 0.05 was considered statistically significant, and all tests were two-tailed tests.

[0048] Example 1 This embodiment aims to screen cfDNA target sequences and design amplification primer sets and probes.

[0049] This study focused on the sequencing analysis of *Basilaria marneffei* cfDNA. First, *Basilaria marneffei* strain AF293 was used to intervene in immune cells to establish an in vitro *Basilaria marneffei* infection model in immune cells. Then, cfDNA was extracted, a library was constructed, and sequencing was performed. Strict quality control was applied to the raw sequencing data. Subsequently, a reference index was constructed based on the *Basilaria marneffei* whole genome sequence. Clean reads were compared with a full-species nucleic acid sequence library using BLAST to screen for cfDNA fragments specifically matching this bacterium. After removing repetitive fragments, limiting sequence size (40-166 bp), removing host background interference, and performing bioinformatics analysis, the top 500 *Basilaria marneffei* cfDNA fragments and their genomic locations were selected by expression level.

[0050] During the analysis, a total of 11,219 different genes were identified, most of which were not found in public databases. After removing fungal homologous genes, the expression levels of the remaining genes were ranked, revealing that the highest expression fragment sequence number reached 95,313, with an average sequence number of 203.265. Gene abundance analysis identified three conserved genes from *Bassula marneffei*: TMATCC_006500, TMATCC_001136, and TMATCC_000757. Highly conserved target sequences of 100-400 bp in length from each of these three genes, without clear homology in other fungi, were extracted, as shown in SEQ ID NO. 1-3. Their expression stability and dominant abundance were validated in the aforementioned repeat sequencing experiments, demonstrating their value for primer design.

[0051] The designed amplification primers and signal reporter probes are shown in Table 1, corresponding to SEQ ID NO. 4~12 in the sequence listing.

[0052] Table 2. Target genes, fragment names, and amplification primer sequences of *Bacteroides marneffei* cfDNA.

[0053] Example 2 This embodiment aims to preliminarily verify the reliability of the *Basilaria marneffei* cfDNA amplification primer sequences obtained in Example 1 using qPCR. Eight patients suspected of *Basilaria marneffei* infection first underwent mixed multiplex qPCR testing, using the above three sets of primers and probes to detect the cfDNA target sequence; then, etiological evidence testing was performed, and the patients were confirmed to be infected with *Basilaria marneffei* or other fungi (Aspergillus or Cryptococcus) and / or special pathogens according to the criteria of the "Guidelines for the Assessment and Treatment of Invasive Pulmonary Fungal Diseases (2025 Edition)".

[0054] The results are as follows Figure 2 and Figure 3 As shown, Figure 2 Four cases showed increased fluorescence intensity; these four cases were... Figure 3 The logarithmic increase of ΔRn indicates the presence of cfDNA target sequences. Pathogen evidence testing results show that these 4 cases were diagnosed with *Bambusa marneffei* infection, while the other 4 cases were infected with other fungi (Aspergillus or Cryptococcus), preliminarily verifying that the above-mentioned cfDNA target sequences and their primers and probes have evaluative capabilities.

[0055] Example 3 To further evaluate the risk assessment efficacy of the mixed multiplex qPCR assay, this embodiment establishes a validation cohort, comparing the mixed multiplex qPCR assay with existing ELISA assays and attempting to establish an evaluation model. The validation cohort included 60 patients suspected of having *Gnaphalium marneffei* infection. These patients first underwent mixed multiplex qPCR and ELISA testing, with the qPCR using the three primer and probe sets obtained in Example 1 to detect cfDNA target sequences. Subsequently, etiological evidence testing was performed, and the *Guidelines for the Assessment and Treatment of Invasive Pulmonary Fungal Diseases (2025 Edition)* were used to confirm whether the patient's infection was *Gnaphalium marneffei* or caused by other fungi (Aspergillus or Cryptococcus) and / or specific pathogens. ROC curve analysis was used to analyze the assessment value of each indicator and its combination, calculating the area under the curve (AUC), 95% confidence interval (95% CI), and determining the optimal assessment cutoff value based on the maximum Youden index. Simultaneously, the corresponding sensitivity, specificity, and accuracy were calculated.

[0056] The results of etiological evidence testing showed that 33 cases in the validation cohort were diagnosed with *Brachys malneffei* infection. The results of mixed multiplex qPCR testing showed that, except for patients with *Brachys malneffei* who showed normal amplification curves, the other infected patients corresponded to fungi and special bacteria, which are two common types of *Brachys malneffei* that may cause confusion in the assessment, and no amplification results were found.

[0057] Risk assessment efficacy ROC curves of the hybrid multiplex qPCR and ELISA assays are shown below. Figure 4 As shown, the blue line represents the mixed multiplex qPCR detection method for cfDNA, with an AUC of 0.738, sensitivity of 0.70, specificity of 0.70, and optimal cutoff value of 37.5; the red line represents the ELISA detection method, with an AUC of 0.677, sensitivity of 0.733, and specificity of 0.50. This demonstrates that although the mixed multiplex qPCR detection method has slightly lower sensitivity than the ELISA method, it offers better detection specificity. Therefore, the qPCR primers described above possess excellent comprehensive detection capabilities.

[0058] Based on the optimal cutoff value of 37.5 for the AUC curve mentioned above, an assessment model was established: if the CT value of the qPCR test result is ≤37.5, the patient can be assessed as having a high risk of *Brachysmus marneffei* infection; if the CT value is >37.5, the risk of *Brachysmus marneffei* infection is low.

[0059] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. The application of a reagent for quantitatively detecting the biomarker cfDNA in the preparation of *Basilella marneffei* infection risk assessment products, characterized in that... The sequence of the biomarker cfDNA is shown in SEQ ID NO.1~3; the risk assessment product determines the risk of *Brachysmus marneffei* infection by detecting the level of the biomarker cfDNA in the patient's biological sample and comparing it with a preset threshold. If the CT value of the biomarker is ≤37.5 during qPCR detection, the risk of *Brachysmus marneffei* infection is relatively high.

2. The application according to claim 1, characterized in that, The reagents for quantitative detection of the biomarker cfDNA include primers and probes: TMATCC_006500_F, TMATCC_006500_R, TMATCC_006500_P, TMATCC_000757_F, TMATCC_000757_R, TMATCC_000757_P, TMATCC_001136_F, TMATCC_001136_R, and TMATCC_001136_P, and the sequences of the primers and probes are shown in SEQ ID NO.4~12.

3. A detection system for assessing the risk of *Cladosporium marneffei* infection, characterized in that, The detection system includes: The detection device is used to detect the level of the biomarker cfDNA in patient biological samples; The comparison device compares the measured level of the biomarker cfDNA with a preset threshold to determine the risk of infection with *Brachysmus marneffei*. The sequence of the marker cfDNA is shown in SEQ ID NO.1~3; The preset threshold is defined as follows: if the CT value of the biomarker is ≤37.5 during qPCR detection, then the risk of *Brachysmus marneffei* infection is relatively high.

Images