Application of system for detecting torque teno virus load in preparation of product for predicting or assisting in predicting condition of hematopoietic stem cell transplantation patient

Abstract

The invention relates to the technical field of medical examination and clinical prediction models. The invention provides an application of a system for detecting torque teno virus load in preparation of a product for predicting or assisting in predicting the condition of a hematopoietic stem cell transplantation patient, and a risk assessment model for predicting the outcome of HSCT patient infection, graft versus host disease, relapse, non-relapse death and the like is established by detecting the dynamic change of torque teno virus load. Therefore, early warning and risk layering of the illness state of the patient before clinical symptoms appear are realized, and the accuracy of patient management and treatment is improved.

Description

Technical Field

[0001] This invention relates to the field of medical testing and clinical prediction modeling technology, and in particular to the application of a system for detecting circovirus load in the preparation of products for predicting or assisting in the prediction of the condition of hematopoietic stem cell transplant patients. Background Technology

[0002] Hematopoietic stem cell transplantation (HSCT) is an important treatment for hematologic disorders, but patients often experience infections, immune reconstitution disorders, and graft-versus-host disease (GVHD) after transplantation. These complications are significant causes of poor prognosis and death. Currently, clinical monitoring of complications mainly relies on clinical symptoms, routine hematological examinations, and imaging methods, but these methods lack sensitivity and predictive ability, often only detecting abnormalities after disease progression, and lacking effective indicators for early prediction and intervention.

[0003] Anelloviridae is ubiquitous in the human body, and its viral load is closely related to the body's immune status. Previous studies have suggested a positive correlation between total anelloviral load and the degree of immunodeficiency. However, there is currently no standardized, systematic method based on anelloviral load to predict disease progression or complication risk in HSCT patients, and there is a lack of operational detection and interpretation procedures in clinical practice.

[0004] Therefore, there is an urgent need to establish a method for predicting the condition of HSCT patients based on circovirus load in order to achieve individualized risk assessment and early intervention. Summary of the Invention

[0005] The purpose of this invention is to provide a system for detecting circovirus load for use in the preparation of products for predicting or assisting in the prediction of the condition of hematopoietic stem cell transplant patients.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of a system for detecting circovirus load in the preparation of products for predicting or assisting in the prediction of the condition of hematopoietic stem cell transplant patients.

[0007] Preferably, the system for detecting circovirus load includes a set of primers and probes for circovirus and a set of primers and probes for internal reference genes.

[0008] Preferably, the circovirus primer and probe kit includes circovirus forward primer sequence 1 as shown in SEQ ID NO.1, circovirus forward primer sequence 2 as shown in SEQ ID NO.2, circovirus reverse primer sequence 1 as shown in SEQ ID NO.3, and circovirus probe sequence as shown in SEQ ID NO.4.

[0009] Preferably, the internal reference gene primer and probe set includes the internal reference gene forward primer sequence as shown in SEQ ID NO.5, the internal reference gene reverse primer sequence as shown in SEQ ID NO.6, and the internal reference gene probe sequence as shown in SEQ ID NO.7.

[0010] Preferably, the 5' end of the thin loop virus probe sequence is labeled with a FAM fluorescent group, and the 3' end is labeled with an NFQ-MGB quencher group.

[0011] Preferably, the 5' end of the internal reference gene probe sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with an NFQ-MGB quencher group.

[0012] The present invention also provides products for predicting or assisting in the prediction of the condition of hematopoietic stem cell transplant patients, including the system for detecting circovirus load.

[0013] By adopting the above technical solution, the present invention has the following beneficial effects: This invention selects TTV as a living biomarker of immune function, collects patient plasma at predetermined time points before and after transplantation, and uses specific primers and probes to perform qPCR quantification of TTV and the internal reference gene HPRT1 (instead of single-time point or simple qualitative detection) to construct a TTV viral load characteristic reflecting the body's immune reconstitution status. This characteristic is then combined with clinical indicators to establish a risk assessment model for predicting outcomes such as infection, graft-versus-host disease (GVHD), relapse, and non-relapse mortality in HSCT patients. The statistical correlation of the above model is concretized into operable interpretation rules, thereby achieving early warning of the patient's condition before the appearance of clinical symptoms, improving the accuracy of patient management and treatment, and effectively solving the problem of insufficient clinical prediction methods for HSCT patients.

[0014] The prediction method described in this invention does not use any complex machine learning models. It can achieve personalized and accurate early risk prediction (predicting patient risk 1-2 weeks before clinical symptoms) based solely on correlation analysis and threshold rules. At the same time, it is easy to operate and provides quantitative indicators for exploring the relationship between immune status and viral community.

[0015] This invention followed up subjects for one year, starting 7 days before transplantation. Results showed that within a certain time window, a rapid increase in TTV viral load (≥10-fold but <100-fold) compared to the previous time point, with a significant positive correlation to symptom scores, was classified as a Level 1 warning point. A sharp increase in TTV viral load (≥100-fold) compared to the previous time point, with a significant positive correlation to symptom scores, was classified as a Level 2 warning point; both were considered "condition deterioration warning periods." When TTV viral load remained at a low level (TTV undetectable or Ct ≥ Ct_L (Ct_L corresponding to the LLOQ / LOD detection system, ranging from 36-38), and the difference between two consecutive tests was ΔCt > -3.3 (no ≥10-fold increase), the patient was considered to be in a relatively stable or low-risk state. Based on these results, the follow-up frequency could be adjusted, and infection monitoring strengthened. Attached Figure Description

[0016] Figure 1 The paired statistical results of the "Ct-symptom score" of 36 subjects in Example 3 of this invention ( Figure 1 The blue line represents the change of log10 (viral load) or Ct value over time, and the red line represents the change of symptom score over time; the horizontal axis represents the number of weeks of follow-up for the corresponding subjects within one year. Detailed Implementation

[0017] This invention provides an application of a system for detecting circovirus load in the preparation of products for predicting or assisting in the prediction of the condition of hematopoietic stem cell transplant patients.

[0018] In this invention, the system for detecting circovirus load includes a complete set of primers and probes for circovirus and a complete set of primers and probes for internal reference genes.

[0019] In this invention, the internal reference gene is the HPRT1 gene.

[0020] In this invention, the circovirus primer and probe kit includes a circovirus forward primer sequence 1, a circovirus forward primer sequence 2, a circovirus reverse primer sequence 1, and a circovirus probe sequence. The circovirus forward primer sequence 1 is shown in SEQ ID NO.1, specifically 5'-ACWKMCGAATGGCTGAGTTT-3'; the circovirus forward primer sequence 2 is shown in SEQ ID NO.2, specifically 5'-RGTGRCGAATGGYWGAGTTT-3'; the circovirus reverse primer sequence 1 is shown in SEQ ID NO.3, specifically 5'-CCCGAATTGCCCCTTGA-3'; and the circovirus probe sequence is shown in SEQ ID NO.4, specifically 5'-GGGCGGGTGCCGAAGGTGAG-3'.

[0021] In this invention, the circovirus forward primer sequence 1 and the circovirus forward primer sequence 2 are preferably used in equimolar amounts.

[0022] In this invention, the 5' end of the thin circular virus probe sequence is labeled with a FAM fluorescent group, and the 3' end is labeled with an NFQ-MGB quenching group.

[0023] In this invention, the reference gene primer and probe kit includes a reference gene forward primer sequence, a reference gene reverse primer sequence, and a reference gene probe sequence. The reference gene forward primer sequence is shown in SEQ ID NO. 5, specifically 5'-GTCACTCCACTCCCATGTC-3'; the reference gene reverse primer sequence is shown in SEQ ID NO. 6, specifically 5'-GTTCTCTGGGAACTCACCTC-3'; and the reference gene probe sequence is shown in SEQ ID NO. 7, specifically 5'-TCTGGCCCTAGTCTCAGACCTTCC-3'.

[0024] In this invention, the 5' end of the internal reference gene probe sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with an NFQ-MGB quencher group.

[0025] In this invention, highly sensitive and broad-spectrum quantitative detection of TTV is achieved through specific primers and probes, and the reliability of the HPRT1 internal reference system is verified. This is the foundation for achieving "quantitative and comparable" results. Compared with existing methods for qualitative or semi-quantitative detection of TTV, this invention provides a standardized and reproducible load determination scheme.

[0026] The present invention also provides products for predicting or assisting in the prediction of the condition of hematopoietic stem cell transplant patients, including the system for detecting circovirus load.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1

[0029] (a) Selection of subjects

[0030] Patients who are planning to receive or have already received HSCT were selected as subjects, specifically including: 1. Patients who meet the indications for HSCT and are scheduled to undergo autologous or allogeneic hematopoietic stem cell transplantation; 2. Patients aged ≥18 years; 3. Patients with complete transplantation-related data and planned follow-up information; 4. Patients who agree to have plasma samples collected at multiple time points before and after transplantation according to the protocol of this invention.

[0031] Patients with prior solid organ transplantation, those with severe primary immunodeficiency such as HIV, and those who died or were lost to follow-up in the early post-transplant period due to non-infectious causes were excluded. This approach avoids serious confounding factors, ensuring that subsequent dynamic changes in total body volume (TTV) accurately reflect the immune reconstitution process after HSCT, providing a reliable basis for disease prediction.

[0032] (II) Plasma Sample Collection and Processing

[0033] Peripheral venous blood should be collected at the following time points: 7-0 days before transplantation; at least twice a week after transplantation, on Mondays and Thursdays (continuous monitoring for one year is recommended in principle).

[0034] Collect 2-4 mL of venous blood into an EDTA-K2 anticoagulant tube each time. Within 2 hours after blood collection, centrifuge at approximately 1600 g for 10 min, collect the supernatant plasma, and then centrifuge at approximately 16000 g for 10 min to remove cell debris. Aliquot the supernatant plasma into 0.5 mL tubes and store at -80℃.

[0035] This step involves obtaining high-quality plasma samples at multiple time points to construct a "time series" of TTV load. It not only examines the level at a specific point in time but also focuses on dynamic trends, providing time-dimensional information for subsequent prediction models.

[0036] (III) Viral DNA Extraction

[0037] Collect 200 μL of plasma and extract total DNA using a PureLink™ Viral RNA / DNA Mini Kit (Silica Column Method), Invitrogen / Life Technologies, with an elution volume of 50 μL. A blank extraction control was included for each batch to monitor for contamination.

[0038] This step enriches the cell-free viral DNA in the plasma and removes proteins and inhibitors to ensure the sensitivity and specificity of subsequent amplification.

[0039] (iv) qPCR quantitative detection of TTV and HPRT1

[0040] 1. Primer and probe design

[0041] To achieve broad-spectrum detection of different Anelloviridae subtypes, this invention designs a primer and probe set based on a conserved region of Anelloviridae (the conserved sequence region ORF1 from position 589 to 2901 within the 5' untranslated region (5'UTR) of the Anelloviridae genome). The set includes a pair of forward primers, a reverse primer, and a TaqMan probe. The Anelloviridae genome is Torque teno virus 1, isolate TA278 (GenBank / INSDC accession: AB008394).

[0042] The forward primer mixture consists of circovirus forward primer sequence 1 (atf1) as shown in SEQ ID NO.1 and circovirus forward primer sequence 2 (atf2) as shown in SEQ ID NO.2; atf1 and atf2 are mixed in equimolar amounts as a forward primer mixture to improve the coverage of different TTV genotypes.

[0043] The reverse primer is the loop virus reverse primer sequence 1 (atr) as shown in SEQ ID NO.3; the TaqMan probe is the loop virus probe sequence (atp) as shown in SEQ ID NO.4, with the 5' end labeled with the FAM fluorescent group and the 3' end labeled with the NFQ-MGB quencher group.

[0044] Simultaneously, a primer and probe set for the internal control HPRT1 gene was designed. By simultaneously detecting TTV and HPRT1, it is possible to determine whether the DNA in the sample is sufficient and whether there is amplification inhibition, thereby ensuring the reliability of TTV quantification results.

[0045] The primer and probe set for the internal reference HPRT1 gene includes the forward primer sequence (icf) shown in SEQ ID NO.5, the reverse primer sequence (icr) shown in SEQ ID NO.6, and the probe sequence (icp) shown in SEQ ID NO.7. The 5' end of the probe sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with an NFQ-MGB quencher group. Specific sequences are shown in Table 1.

[0046] Table 1 Primer and probe sequence information

[0047] 2. qPCR amplification

[0048] Taking singlet qPCR reaction as an example, each well contains a 25 μL reaction system including: 2× Probe qPCR Mix: 12.5 μL; Forward primer (10 μM): 0.5 μL (atf1 / atf2 mixture for TTV detection, icf for HPRT1 detection); Reverse primer (10 μM): 0.5 μL (atr for TTV detection, icr for HPRT1 detection); Probe (10 μM): 1.0 μL (TTV is ATP, HPRT1 is ICP); ROX Ⅱ (100×): 0.25 μL; Sample DNA: 2.0 μL; Finally, add nuclease-free water to bring the volume to 25 μL.

[0049] Based on the above qPCR reaction system, each sample was prepared in 2-3 replicates, and a DNA-free control and a positive control were included (plasma from patients with TTV-positive sequencing results was used as the positive control). Amplification was then performed on a real-time fluorescence PCR instrument using the following program: 95℃ pre-denaturation for 2-3 min; 95℃ for 10 s, 60℃ for 30 s, 45 cycles; 72℃ for 30 s. Fluorescence signals were collected and melting curves were plotted for analysis.

[0050] Using a standard containing a known copy number of TTV target sequence (10) 2 -10 8 A standard curve was prepared using copies / mL, and the TTV copy number of the sample was calculated and converted into the load per mL of plasma.

[0051] (V) Construction of TTV viral load characteristics

[0052] Based on TTV load data at various time points, the following features were extracted for each patient, including but not limited to: 1. Absolute load at each time point; 2. fold change or change in load relative to baseline; 3. Maximum load during the observation period and its occurrence time; 4. Area under the load-time curve (AUC) or mean load; 5. Degree of load fluctuation (standard deviation or coefficient of variation, etc.). This transforms the raw time-series data into biologically and clinically significant quantitative features, enabling TTV load to reflect not only the level at a specific time point but also the dynamic process of immune reconstitution.

[0053] (vi) Correlation analysis and disease assessment

[0054] Clinical indicators were obtained, and then for each patient, in the order of sample collection, the TTV log10 (fold change) at the same time point was paired with the symptom score, as shown in the attached figure. Figure 1 As shown. Spearman rank correlation or Pearson correlation analysis was used to calculate the correlation coefficient. r (or ρ) and p Value, when r >0 (preferred) r ≥0.5) and p A TTV load of <0.05 is considered to be significantly positively correlated with symptom exacerbation. For multiple patient cohorts, the correlation coefficients for each patient can be aggregated, and the proportion of patients with significant correlations can be calculated to verify the stability of TTV as a disease biomarker.

[0055] The clinical indicators include one or more of the following: 1. Patient clinical symptom scores (such as infection symptom scores, GVHD rash scores, diarrhea scores, etc.); 2. Laboratory indicators (inflammatory markers, organ function indicators); 3. Complication grading (such as CTCAE grading, GVHD grading), etc.

[0056] Risk assessment specifications: Within a certain time window, if the TTV load at a later time point increases by ≥10 times but <100 times compared to the previous time point (rapid increase), and shows a significant positive correlation with the symptom score, then that time point is classified as a Level 1 warning. If the TTV load at a later time point increases by ≥100 times compared to the previous time point (sharp increase), and shows a significant positive correlation with the symptom score, then that time point is classified as a Level 2 warning. Both are considered "condition deterioration warning periods." When the TTV load remains at a low level, i.e., TTV is undetectable or Ct ≥ Ct_L (Ct_L corresponding to the LLOQ / LOD detection system, ranging from 36 to 38), and ΔCt > -3.3 between two consecutive tests (no ≥10-fold increase), then the patient is considered to be in a relatively stable or low-risk state.

[0057] Example 2

[0058] (I) Study Subjects and Follow-up Design

[0059] Thirty-seven patients who underwent hematopoietic stem cell transplantation (HSCT) at our hospital (Peking University People's Hospital) were selected. One patient was excluded due to insufficient hospital stay and inadequate available plasma samples. Ultimately, 36 recipients were included as the subjects of this study.

[0060] During routine clinical testing, residual plasma samples were collected from these 36 patients before hospitalization and their first discharge. Multiple plasma tubes collected at the same time point were mixed in equal volumes and used as the test samples for that time point. A total of 756 mixed plasma samples were obtained (approximately 10-30 samples per patient, with an average of approximately 21 samples).

[0061] (II) Quantification methods for symptom recording and "symptom scoring"

[0062] To objectively quantify the severity of the disease at each time point, this embodiment establishes a binary scoring system (present / absent) based on the nine clinical manifestations shown in Table 2. For each mixed plasma sample, the presence or absence of each of the nine manifestations is determined: 1 point is awarded for each present symptom, and 0 points are awarded for each absent symptom.

[0063] The symptom score at each time point is the sum of the scores of the above 9 items, with a theoretical range of 0-9 points, used to quantify the patient's overall clinical symptom burden at that time point.

[0064] Table 2 Clinical Standards for the Binary Scoring System

[0065] In this invention, the "infection diagnostic criteria" described in Table 1 refer to meeting any of the following: (1) Etiologically confirmed infection: positive blood culture or sterile body fluid / tissue culture, or nucleic acid detection of a clear pathogen in blood or corresponding clinical specimens and consistent with clinical manifestations; (2) Clinical diagnosis of infection: The presence of infection-related symptoms / signs and / or imaging findings suggestive of an infection focus, and the diagnosis made by a clinician and the initiation or escalation of anti-infective treatment accordingly; (3) For patients with neutropenia, those who meet the definition of fever in febrile neutropenia may be included in the infection / febrile event (or counted as fever of unknown cause).

[0066] Statistical results showed that most of the 36 patients experienced 2-4 symptoms at some stage of the disease course; some patients experienced up to 6 symptoms simultaneously, corresponding to a symptom score of 6 (see...). Figure 1 ).

[0067] (III) Anelloviridae Ct value detection

[0068] All 756 mixed plasma samples were subjected to nucleic acid extraction according to the method in Example 1, and Anelloviridae was detected by real-time fluorescent qPCR using specific primers and probes.

[0069] Primers and probes were designed to target the conserved 5' non-coding region of Anelloviridae (ORF1 positions 589-2901), specifically the Torque teno virus 1 genome, isolate TA278 (GenBank / INSDC accession: AB008394). The forward / reverse primers used were the universal TTV primers atf1, atf2, and atr. Probes were designed to target conserved Anelloviridae sequences (5' FAM, 3' NFQ-MGB) for fluorescence signal detection. Specific sequences are shown in Table 1.

[0070] Each sample was prepared with 2-3 technical replicates, and the average Ct value was taken as the Anelloviridae Ct value at that time point. The lower the Ct value, the higher the Anelloviridae load in the plasma.

[0071] (iv) Data pairing and statistical analysis methods

[0072] For each patient: 1. List all sampling time points in chronological order; 2. Pair the Anelloviridae Ct value at the same time point with the corresponding symptom score to form time series data; 3. Plot two line graphs (blue line represents the change of log10 (viral load) or Ct value over time; red line represents the change of symptom score over time) (see... Figure 1 ).

[0073] Spearman rank correlation analysis was then used to calculate the correlation coefficient between Anelloviridae Ct values ​​and symptom scores for each patient. r and p value.

[0074] like p A Ct value <0.05 is considered statistically significant in relation to the patient's symptom score. Since the Ct value is inversely proportional to viral load, [the following is a separate, unrelated statement:] ... r Negative values ​​and p A score <0.05 indicates that the lower the Ct value (the higher the viral load), the higher the symptom score and the more severe the disease.

[0075] (v) Results Data

[0076] Analysis of 756 pairs of paired Ct-symptom score data from 36 participants yielded the following results: 1. Overall Relevance Of the 36 patients, 20 showed a high correlation between the trend of their Anelloviridae Ct values ​​and their symptom scores, as demonstrated by Spearman correlation analysis. pAll values ​​were <0.05. For these 20 patients, the correlation coefficient between Ct values ​​and symptom scores was... r Most values ​​were negative, with a median of approximately -0.58 (interquartile range -0.71 to -0.45), indicating that lower Ct values ​​correlated with a heavier symptom burden. For the remaining 16 patients, the correlation was not statistically significant in some cases due to milder overall symptoms and limited variation.

[0077] 2. Dynamic changes in representative patients

[0078] During the peak of the disease course, patient C(P-10) had a symptom score as high as 6, while the Anelloviridae Ct value dropped to about 20-22, a decrease of about 5 cycles from baseline. Subsequently, after intensive treatment, the symptom score gradually dropped to 0-1, and the Ct value increased to about 30, indicating that the decrease in viral load was consistent with the symptom relief.

[0079] Patient R(P-16) also had a symptom score of 6 during the period of severe diarrhea, fever, and rash, with the corresponding Ct value decreasing from 30 to approximately 21; after treatment, the Ct value rebounded and the symptom score decreased. Spearman correlation analysis p <0.01.

[0080] Similar phenomena were observed in several other patients: the Ct value initially decreased significantly, then gradually increased as the clinical condition improved, and the peaks and troughs of the Ct curve and the symptom score curve largely coincided on the time axis (see...). Figure 1 ).

[0081] 3. Prognostic observations

[0082] During the follow-up, it was observed that some patients showed a continuous downward trend in Ct value before molecular relapse; patients with better prognosis showed an overall gradual upward trend in Ct value (decreasing load); and a small number of patients maintained a low Ct value for a long time after transplantation, which may indicate a potentially high-risk state, even though clinical symptoms were not obvious at the time.

[0083] In summary, the "symptom score" constructed based on the above nine objective indicators can effectively quantify the disease severity of HSCT patients at each time point. By detecting Anelloviridae using qPCR and recording Ct values, combined with time series analysis and Spearman correlation tests, it was demonstrated that there is a strong correlation between the trend of Ct value changes and clinical symptom burden; the lower the Ct (the higher the viral load), the more numerous and severe the symptoms experienced by the patient. This correlation reached statistical significance in 20 out of 36 patients, indicating that the method proposed in this invention, "using the dynamic change of Anelloviridae Ct value (dynamic change of Anelloviridae viral load) as an indicator for monitoring HSCT disease," has good universality and clinical application value.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of a system for detecting a circovirus load in the preparation of a product for predicting or assisting in predicting the condition of a patient undergoing hematopoietic stem cell transplantation.

2. Use according to claim 1, characterized in that, The system for detecting a circovirus load comprises a circovirus complete primer probe set and an internal reference gene complete primer probe set.

3. Use according to claim 2, characterized in that, The circovirus complete primer probe set comprises a circovirus forward primer sequence 1 as shown in SEQ ID NO. 1, a circovirus forward primer sequence 2 as shown in SEQ ID NO. 2, a circovirus reverse primer sequence 1 as shown in SEQ ID NO. 3, and a circovirus probe sequence as shown in SEQ ID NO. 4; The internal reference gene complete primer probe set comprises an internal reference gene forward primer sequence as shown in SEQ ID NO. 5, an internal reference gene reverse primer sequence as shown in SEQ ID NO. 6, and an internal reference gene probe sequence as shown in SEQ ID NO.

7.

4. Use according to claim 3, characterized in that, The 5' end of the circovirus probe sequence is labeled with a FAM fluorescent group, and the 3' end is labeled with a NFQ-MGB quenching group. The 5' end of the internal reference gene probe sequence is labeled with a VIC fluorescent group, and the 3' end is labeled with a NFQ-MGB quenching group.

5. A product for predicting or aiding in the prediction of the condition of a patient undergoing hematopoietic stem cell transplantation, characterized in that, The system for detecting a circovirus load as claimed in claim 1.

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