Monitoring of viral sequences in virus-infected tissues

Abstract

The present invention provides a means for detecting viral sequences in virus-infected tissue by specific amplification of the viral sequence. [Solution] A method for monitoring viral sequences 14 in virus-infected tissue, wherein cell-free DNA in blood 25 taken from a host 10 is used as a template, and PCR is performed using a pair of viral primers 26 assigned to the viral genome sequence and individualized primers 27 assigned to the host 10 genome sequence to detect an amplified product including the boundary between the viral genome 11 and the host genome 12, wherein the individualized primers 27 are assigned to sequences 16 near the site 13 in which the virus is incorporated on the host 10 genome sequence, and the site 13 in which the virus is incorporated on the genome sequence has been identified from a sample 23 taken from the host 10 before monitoring 24.

Description

[[Technical Field]]

[0001] The present invention relates to monitoring viral sequences in virus-infected tissues. [[Background Art]]

[0002] Patent Document 1 discloses a method for screening a pathogen-associated disorder in a subject, wherein a first assay comprises characterizing cell-free nucleic acid molecules from a pathogen in a biological sample of the subject; and based on the characterized features, a second assay screens for the pathogen-associated disorder in the subject. Patent Document 2 and Non-Patent Document 1 disclose methods for determining the integration site of viral nucleic acid into a host genome.

[0003] The abstract of Non-Patent Document 2 discloses that virus-host chimeric DNA (vh-DNA) generated from the integration junction of hepatitis B virus (HBV) in the chromosomes of HCC is used as a circulating biomarker. vh-DNA was quantified by digital PCR (ddPCR) assay in plasma samples collected before surgery and 2 months after surgery.

[0004] Lines 10 to 5 from the bottom of the right column on page 5870 of Non-Patent Document 3 describe that using primers and TaqMan probes specific for HPV16 E7, HPV18 E7, or virus-cell junctions, HPV16, HPV18, or HPV integration sites were detected from serum by digital droplet PCR. The abstract of the same document shows that circulating HPV E7 gene is a more sensitive tumor marker than HPV integration sites in serum. [[Prior Art Documents]] [[Patent Documents]]

[0005] [[Patent Document 1]] Japanese Unexamined Patent Publication No. 2022-527316 [[Patent Document 2]] Japanese Unexamined Patent Publication No. 2015-133957

Non-licensed literature

[0006] [Non-licensed document 1] Weiyang Li, Xi Zeng, Nikki P. Lee, Xiao Liu, Shengpei Chen, Bing Guo, Shang Yi, Xuehan Zhuang, Fang Chen, Guan Wang, Ronnie T. Poon, Sheung Tat Fan, Mao Mao, Yingrui Li, Songgang Li, Jun Wang, JianWang, Xun Xu, Hui Jiang, Xiuqing Zhang, “An efficient method to detect HBV integration using low coverage sequencing”, [online], Genomics, Volume 102, Issue 4, 2013, Pages 338-344, [retrieved on 2024-04-03]<URL: https: / / doi.org / 10.1016 / j.ygeno.2013.07.002>

[0007] [Non-licensed document 2] Li, Chiao‐Ling; Ho, Ming‐Chih; Lin, You‐You; Tzeng, Sheng‐Tai; Chen, Yun‐Ju; Pai, Hsin‐Yung; Wang, Ya‐Chun; Chen, Chi‐Ling; Lee, Yu‐Hsin; Chen, Ding‐Shinn; Yeh, Shiou‐Hwei; Chen, Pei‐Jer. Cell‐Free Virus‐Host Chimera DNA From Hepatitis B Virus Integration Sites as a Circulating Biomarker of Hepatocellular Cancer. Hepatology 72(6):p 2063-2076, December 2020. | DOI: 10.1002 / hep.31230, [retrieved on 2024-10-22],<URL: https: / / doi.org / 10.1002 / hep.31230>

[0008]

Table 3

[0009] The inventors attempted to monitor viral sequences in virus-infected tissue by performing PCR using cell-free DNA (cfDNA) in the blood as a template. However, PCR amplifies not only cell-free DNA in the blood but also the genomic DNA of free viruses in the blood. This invention provides a means for detecting viral sequences in virus-infected tissue through specific amplification of the viral sequence. [Means for solving the problem]

[0010] <1> A method for monitoring viral sequences in virus-infected tissue, Using cell-free DNA from the host's blood as a template, Viral primers assigned to the viral genome sequence, Individualized primers assigned to the host genome sequence, Quantitative PCR was performed on the following groups. The amplification product, which includes the boundary between the viral genome and the host genome, is detected. Here, The individualized primer is assigned to a sequence near the site on the host genome sequence in which the virus is incorporated. The site on the genome sequence in which the virus is incorporated has been identified from a sample taken from the host before monitoring. method.

[0011] <2> The aforementioned sample was obtained from tissue that had become diseased due to infection with the aforementioned virus. The method described above.

[0012] <3> The sites identified from the diseased tissue are narrowed down based on the condition that they are also identified as sites where the virus is incorporated from cell-free DNA in blood collected from the host before monitoring. The method described above.

[0013] <4> The aforementioned sample is cell-free DNA from blood. that said site is derived from a tissue affected by a disease caused by infection with said virus is identified based on the fact that said host was diagnosed as having said disease when blood was collected before monitoring, The method according to any one of the above.

[0014] <5> The virus primer is specific to a viral species or subtype identified by genotyping of free virus collected from the periphery of the virus-infected tissue before monitoring, The method according to any one of the above.

[0015] <6> The free virus is collected from the host before collecting the sample from the host, The method according to any one of the above.

[0016] <7> The disease occurring in the virus-infected tissue is cancer, The method according to any one of the above.

[0017] <8> The virus is a DNA virus, The method according to any one of the above.

[0018] <9> The disease occurring in the virus-infected tissue is cervical cancer, the virus is human papillomavirus (HPV), and the tissue is cervical tissue, The method according to any one of the above.

[0019] <10> The vicinity of the site where the virus is integrated refers to a range that can be amplified by said PCR starting from the site where said virus is integrated, The method according to any one of the above.

[0020] <11> The presence or absence of detection of the amplification product or the detection amount of the amplification product is provided as data for diagnosis of a disease occurring in the virus-infected tissue, The method according to any one of the above.

[0021] <12> A reagent for monitoring viral sequences in virus-infected tissue, It contains viral primers assigned to the viral genome sequence, In that usage, Using cell-free DNA from the host's blood as a template, The aforementioned virus primer, Individualized primers assigned to the host genome sequence, PCR was performed in the group. The amplification product, which includes the boundary between the viral genome and the host genome, is detected. Here, The individualized primer is assigned to a sequence near the site on the host genome sequence in which the virus is incorporated. The site in which the virus is incorporated has been identified from a sample taken from the host before monitoring. reagent.

[0022] <13> In that usage, When the sample was collected from the host before monitoring, the host was suffering from a disease caused by infection with the virus. If the detected amount of the amplification product exceeds the standard value, the host is diagnosed with a recurrence of the disease. The reagents mentioned above. [Effects of the Invention]

[0023] The present invention provides a means for detecting viral sequences in virus-infected tissue by specific amplification of the viral sequence. [Brief explanation of the drawing]

[0024] [Figure 1] Virus sequence detection flow [Figure 2] Scheme for detecting viral sequences [Figure 3] Venn diagram for narrowing down the insertion site [Figure 4] Flowchart for calculating Cq value [Figure 5] Specific procedure for calculating Cq value Mode for Carrying Out the Invention

[0025] <Detection of Viral Sequences by PCR>

[0026] Figure 1 shows a flow of viral sequence detection. In step S01, an infection marker for a viral infectious disease is identified. In step S02, a primer pair assigned to the infection marker is selected. In step S03, PCR is performed using the primer pair to detect a viral sequence causing infection associated with the disease. If necessary, diagnostic data is prepared from the detection results in step S04. The diagnostic data is provided to a person who manages the disease. In the present embodiment, the term viral sequence refers to a full-length or partial sequence of a viral sequence integrated into the host genome.

[0027] Figure 2 shows a scheme for viral sequence detection. In the present embodiment, monitoring 24 of viral sequences in tissues of a virus-infected host 10 is performed. More specifically, a viral genome 11 integrated into a host genome 12 of the host 10 is detected.

[0028] In Figure 2, when it is diagnosed in diagnosis 19 that the host 10 has developed a disease caused by viral infection, treatment 22 and monitoring 24 are planned. At the time of treatment 22, blood 20 and / or a pathological specimen 23 is collected from the host 10 suspected of having a viral infection. The blood 20 and the pathological specimen 23 may be collected during treatment 22, or may be collected during an examination before treatment 22. The treatment 22 may involve therapy. The therapy may be surgery, or may be chemical, biological or physical therapy. The treatment 22 does not have to involve therapy, and may be limited to collection of the blood 20 and the pathological specimen 23. The pathological specimen 23 is collected from a tissue that exhibits symptoms due to viral infection. For example, the tissue may be a cervix exhibiting symptoms of cervical cancer.

[0029] In Figure 2, a personalized infection marker 15 is identified from the pathological specimen 23 for the host 10. The pathological specimen 23 may be FFPE (formalin fixed paraffin embedded). The pathological specimen 23 may be replaced with cells obtained from a cytological examination of diseased tissue, such as a scraping cytology. Here, the infection marker 15 can also be considered a disease marker. For example, if the disease is a tumor, the infection marker 15 can also be considered a tumor marker.

[0030] In Figure 2, the infection marker 15 spans both the viral sequence 14 and the neighboring sequence 16. The viral sequence 14 is the sequence resulting from the integration of the viral genome 11 into the insertion site 13 on the host genome 12. The viral sequence 14 is sometimes called a provirus. The neighboring sequence 16 is the sequence adjacent to the insertion site 13 where the viral sequence 14 is located. The insertion site 13 is identified from a sample taken from the host 10. In the figure, it is identified from at least one of blood 20 and a pathological specimen 23. In this embodiment, sequences other than the neighboring sequence 16 on the host genome 12 are called non-neighboring sequences 17.

[0031] In Figure 2, the pair of infection marker 15 and neighboring sequence 16 occurs both upstream and downstream of the host genome 12. At least one of them is selected as infection marker 15. In the figure, the infection marker 15 on the right is used as a representative example.

[0032] In Figure 2, the individualization of infection markers 15 is due to the random determination of the insertion site 13 on the host genome 12 into which the viral genome 11 is incorporated. Thus, the insertion site 13 is unknown. In other words, while the viral sequence 14 is a known sequence, the sequences of neighboring sequences 16 are unknown. Even if the sequence information of neighboring sequences 16 is identified, it is unknown whether that sequence information belongs to an upstream or downstream neighboring sequence 16 of the viral sequence 14, or which orientation it represents. Therefore, a separate process is required to identify this information. This process will be described later.

[0033] In Figure 2, monitoring 24 is performed after the procedure 22. Monitoring 24 is provided as a liquid biopsy test by taking blood from the host 10. During the monitoring 24 period, blood 25 is taken from the host 10 multiple times as needed. The blood 25 contains cell-free DNA 30. Cell-free DNA 30 consists of fragments of the host genome 12. Such fragments may contain viral sequences 14. If the disease is a tumor, cell-free DNA 30 is also called ctDNA (circulating tumor DNA).

[0034] In Figure 2, PCR is performed using cell-free DNA 30 as a template with pairs of viral primer 26 and individualized primer 27. Viral primer 26 is assigned to viral sequence 14. Individualized primer 27 is assigned to host genome 12, more specifically to neighboring sequence 16. The amplification product includes the boundary between viral sequence 14 and neighboring sequence 16. This allows PCR to target infection marker 15.

[0035] In Figure 2, the range of the vicinity of insertion site 13 related to neighboring sequence 16 depends on the length that can be amplified by PCR. Neighboring sequence 16 is greater than 0 kbp (kilobase pair) and less than or equal to 20 kbp, starting from insertion site 13. Neighboring sequence 16 is one of the following ranges starting from insertion site 13: 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 kbp (kilobase pairs). However, since the amplification product also extends over viral sequence 14, the range of neighboring sequence 16 is limited to account for that portion.

[0036] As shown in Figure 2, the blood 25 also contains free viruses. The free viruses include the viral genome 11. Here, separately from this embodiment, we assume that PCR is performed using forward primer 31 and reverse primer 32 that target the inside of the viral sequence 14, as shown in Figure 2. In this case, not only the cell-free DNA 30 but also the viral genome 11 derived from the free viruses acts as a template. Therefore, the amplification product is derived from both. Furthermore, viral sequences incorporated into non-neighboring sequences 17 on the host genome sequence, which are not neighboring sequences 16 associated with the infection marker 15, also act as templates. For this reason, it is difficult to specifically detect the infection marker 15 using forward primer 31 and reverse primer 32. In other words, false positives are likely to occur. In contrast, the infection marker 15 can be specifically detected by performing PCR using a pair of viral primer 26 and individualized primer 27. In other words, false positives are less likely to occur.

[0037] As shown in Figure 2, in the PCR detection of the viral sequence in this embodiment, the viral primer 26 is assigned to the infection marker 15, making it specific to the type of virus that caused the infection. Furthermore, since the individualized primer 27 is assigned to the host genome 12, it is specific to the virus in the infected tissue.

[0038] <Identification of infection markers>

[0039] In Figure 2, the identification of the infection marker 15 is performed by identifying the insertion site 13 on the host genome 12 into which the viral genome 11 is incorporated. This identification is performed by obtaining sequence information of neighboring sequences 16. Sequence information of neighboring sequences 16 is collected from diseased tissue or other tissue samples of the host 10 before monitoring 24. For example, it is obtained from blood 20 or pathological specimens 23. Specifically, sequence information of neighboring sequences 16 is obtained before PCR is performed using cell-free DNA 30 as a template during the monitoring 24 period.

[0040] In Figure 2, multiple candidate insertion sites 13 into which the viral genome 11 is incorporated may be identified. Some of these insertion sites 13 may be difficult to detect in the blood as cell-free DNA. Therefore, being identifiable as an insertion site from cell-free DNA may be used as a condition for narrowing down the candidates for insertion site 13.

[0041] Figure 3 is a Venn diagram showing the narrowing down of insertion sites into which the virus is incorporated. The upper circle shows a set of insertion sites 13 identified from diseased tissue, such as pathological specimens 23. The lower circle shows a set of insertion sites identified from cell-free DNA in blood 20. These co-sets indicate that the insertion sites originate from diseased tissue and are present in the bloodstream. Diseased tissue may simply be tissue infected with the virus, regardless of whether it is diseased or not. The infection markers 15 identified from these narrowed-down insertion sites 13 can be suitably used in subsequent PCR monitoring.

[0042] Returning to Figure 2, in embodiments different from those shown in the figure, the insertion site 13 is identified from cell-free DNA in blood, for example, blood 20, collected from the host 10 before monitoring 24. If both the pathological specimen 23 and the blood 20 are available, it is preferable to analyze both to more reliably identify the insertion site 13 or the infection marker 15. More specifically, since there are multiple insertion sites on the genome, it is possible that some regions of the insertion site are more likely to flow into the blood as cell-free DNA, while others are less likely to become cell-free DNA. Therefore, by analyzing both the blood and the pathological specimen, an insertion site that can be detected from both is selected. This makes it more certain that the set infection marker will actually function as an infection marker. Also, in certain cases, information on sites identified from diseased tissue is not used. For example, for a host 10 that does not undergo surgery, a pathological specimen 23 may not be collected in order to avoid invasiveness to the body as much as possible, and only the obtained blood 20 may be analyzed. The fact that the insertion site 13 originates from diseased tissue of the host 10 is identified based on the diagnosis that the host 10 was diseased when the blood was collected before monitoring 24. To confirm that cell-free DNA originates from diseased tissue and not from other infected tissues, it is preferable to confirm that infection markers identified from blood are also present in pathological specimens.

[0043] The procedure for identifying the insertion site 13 is described in Figure 2. First, sequence information of an unknown neighboring sequence 16 is obtained. In this process, DNA from the host genome 12 in the collected sample, i.e., the pathological specimen 23, or cell-free DNA from the blood 20 is extracted. Next, sequence information of the neighboring sequence 16 is obtained using any means. When obtaining the sequence information of the neighboring sequence 16, sequence information of the terminal portion of the viral sequence 14 is also obtained. This allows for the identification of whether the neighboring sequence 16 is adjacent to the upstream or downstream portion of the viral sequence 14, and which orientation it has.

[0044] In Figure 2, one of the following methods is used to obtain sequence information of neighboring sequences 16: Alu-PCR, inverse PCR, next-generation sequencing (NGS), probe-based methods, or other methods. In Alu-PCR and inverse PCR, the sequence of the amplified product is determined. In NGS, the sequence of the extracted DNA is comprehensively determined. Probe-based methods are also called target capture or genome capture. First, the extracted DNA is fragmented. Next, DNA fragments that hybridize with probes assigned to the sequence of the viral genome 11 are collected. Then, the sequence of the collected DNA fragments is determined by NGS or other methods. This allows for the detection of insertion sites 13 that occur randomly on the host genome 12.

[0045] <Selection of primer>

[0046] In Figure 2, the viral primer 26 is assigned to the viral sequence 14. The viral primer 26 may be pre-designed based on the sequence information of the viral genome 11. The design strategy will be described later. The viral primer 26 is complementary to the viral sequence 14 incorporated into the host genome and is a primer that faces outward from the viral sequence 14. The viral primer 26 and the individualized primer 27 may be selected from existing primers. The selection strategy will be described later.

[0047] In Figure 2, each of the viral primers 26 and individualized primers 27 is designed considering the Tm value, GC content, number of bases, and bias between GC bases and AT bases. For example, primer sequence information may be generated by automatic calculation based on the following example design conditions. Consideration may also be given to the generation of primer dimers and the suppression of nonspecific proliferation from other sites on the genome.

[0048] <Examples of primer design conditions> The length of the PCR product is arbitrary, but in one example it ranged from 0.05 kbp to 30 kbp, in another example from 0.075 kbp to 3 kbp, and in yet another example from 0.1 kbp to 0.3 kbp. Primer length: minimum 18 bases, optimal 20 bases, maximum 25 bases • The Tm value ranges from a minimum of 50°C to an optimal of 60°C and a maximum of 63°C. • GC content: maximum 40%, optimal 50%, maximum 60% • The 3' end is limited to G or C. • The number of allowed GC bases in the 5 bases at the 3' end is 3 • The number of allowed single-base repetitions is 3

[0049] In Figure 2, it is preferable that the viral primer 26 hybridizes with the viral sequence 14 at a point between 0.01 kbp and 10 kbp, starting from the boundary between the viral sequence 14 and the neighboring sequence 16. The viral primer 26 hybridizes with the viral sequence 14 at points of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 kbp, starting from the boundary between the viral sequence 14 and the neighboring sequence 16. The viral primer 26 has its 3' end on the side of the neighboring sequence 16 to which the individualized primer 27 is assigned.

[0050] In Figure 2, the individualized primer 27 is assigned to the neighboring sequence 16. The individualized primer 27 is selected based on the sequence information of the neighboring sequence 16. The individualized primer 27 may be newly created. The individualized primer 27 has its 3' end on the side of the viral sequence 14.

[0051] <Types of viruses and types of diseases>

[0052] In Figure 2, the virus possessing the viral genome 11 is a virus that is integrated into the host genome 12. The virus is not limited in any other respect. The disease that occurs in the host 10 due to viral infection is not limited. The type of tissue or cell that the virus infects is not limited.

[0053] In one embodiment shown in Figure 2, the virus having the viral genome 11 is, for example, a DNA virus or an RNA virus. Such a virus is, for example, a single-stranded virus or a double-stranded virus. When the virus having the viral genome 11 is an RNA virus, the viral sequence 14 inserted into the host genome 12 is produced by the reverse transcription of the viral genome 11.

[0054] In one embodiment shown in Figure 2, the disease of the host 10 is cancer. A virus possessing the viral genome 11 infects a tissue, causing cancer in that tissue or other tissues. Examples of cancers include cervical cancer and liver cancer. Viruses possessing the viral genome 11 include human papillomavirus (HPV) and hepatitis B virus (HBV).

[0055] <Strategies for designing and selecting viral primers>

[0056] In Figure 2, the viral primer 26 is useful for detecting the genomic DNA of a specific type of virus from cell-free DNA 30. The design of the viral primer 26 allows for changes in the granularity for detecting different types of viruses. For example, using a sequence that is conserved among many types of viruses as a primer allows for the detection of many types of viruses. Alternatively, using a sequence that is specific to certain types of viruses as a primer allows for the detection of specific types of viruses. Here, a specific type of virus may be a virus of a specific species, or a specific subtype of a virus of a specific species. The subtype may be one that has been previously identified as causing a disease.

[0057] The viral primer may be specific to HPV, for example. The viral primer may be a sequence conserved among HPV subtypes. The viral primer may be a sequence found only in specific HPV subtypes. Examples of specific HPV subtypes include HPV16, HPV52, and HPV18.

[0058] When using sequences specific to certain types of viruses as primers, it is necessary to know the type of virus infecting the host before monitoring. For example, the type of virus can be identified based on the sequence information of the virus sequence 14, which is obtained simultaneously when acquiring the sequence information of the neighboring sequence 16 shown in Figure 2.

[0059] Alternatively, when identifying the infection marker as described above, the type of virus, such as the virus species or subtype, may be identified before monitoring. Starting from the sequence information of such a specific virus type, the insertion site 13 in which the virus is incorporated, the infection marker 15, and the neighboring sequences 16 are identified using the method described above. If a probe is used, the probe sequence may be specific to the virus type.

[0060] Another method for identifying the type of virus is to identify the subtype by genotyping of free viruses collected from the surrounding tissue before monitoring in diagnostic 19, as shown in Figure 2. Free viruses may also be obtained along with cells during cytology, such as swab cytology. For example, free viruses may be collected from the host in diagnostic 19 before taking a sample from the host for marker identification in procedure 22. Based on the subtype identified by genotyping, a decision may be made whether or not to perform procedure 22, or whether or not to choose drug treatment or surgical treatment, or whether or not to perform monitoring. Genotyping may be performed as an integrated diagnosis in combination with other diagnostic steps by the physician. As mentioned above, the information on the virus species or subtype obtained by genotyping is also used to improve the specificity of viral primers in monitoring.

[0061] The genotyping method is not limited. For example, DNA sequencing, SSCP (Single Strand Conformation Polymorphism), RFLP (Restriction Fragment Length Polymorphism), PCR, AFLP (Amplified Fragment Length Polymorphism), ASO (Allele Specific Oligonucleotide) probes, DNA microarrays, and DNA beads can be used.

[0062] <Creating diagnostic data>

[0063] Returning to Figure 1, after detecting the viral sequence by PCR in step S03, diagnostic data is created from the detection results in step S04. Specifically, diagnostic data is created based on the presence or absence of detection of amplification products or the amount detected. The diagnostic data is provided to the medical manager of the disease of host 10 shown in Figure 2, for example, the attending physician.

[0064] In the example shown in Figure 1, the PCR in step S03 may be quantitative PCR. Quantitative PCR is also called real-time PCR or qPCR. Quantitative PCR preferably uses a method that detects a fluorescent signal. Unlike digital PCR, quantitative PCR uses a bulk mixture of DNA as a template for the reaction. In contrast, digital PCR involves distributing the template DNA one molecule at a time into droplets using limiting dilution before the reaction is carried out.

[0065] Quantitative PCR may be performed using an intercalator such as SYBR Green™ or a hydrolysis probe such as TaqMan™ probe. In quantitative PCR, for example, a threshold value for the Ct value may be set in advance, and detection of the amplification product may be defined as the Ct value falling below the threshold. Alternatively, the detection amount may be obtained by quantifying the amplification product. Furthermore, in step S04, mathematical analysis, statistical analysis, or visual processing such as graphing may be performed based on the detection amount. A medical administrator, such as the attending physician, may diagnose that host 10 has relapsed if the detection amount of the PCR amplification product exceeds the reference value. A medical administrator, such as the attending physician, may diagnose that the treatment is effective if the detection amount of the PCR amplification product does not exceed the reference value. The reference value is set appropriately based on clinical statistical analysis and expert review.

[0066] <Quantitative PCR and Mathematical Analysis>

[0067] In the example shown in Figure 1, quantitative PCR may be used in step S03. Quantitative PCR requires simpler procedures and equipment compared to digital PCR. Therefore, quantitative PCR is suitable for repeated testing during the monitoring period. Furthermore, quantitative PCR is suitable for performing PCR on multiple hosts simultaneously in a single facility, for example, using a multiplex system.

[0068] In step S04 shown in Figure 1, mathematical analysis suitable for detecting the presence of low concentrations of infection markers from the output data of quantitative PCR can be employed. This combination of repeated quantitative PCR testing and improved sensitivity through mathematical analysis can assist medical administrators in their diagnoses.

[0069] Figure 4 shows the flow of calculating the Cq value as an example of mathematical analysis. In the flow, the Cq value of the amplification curve is obtained by performing nonlinear regression to the amplification curve. In this embodiment, the Cq value represents the number of cycles in quantitative PCR in which the signal of the amplified product exceeds the background level.

[0070] In step S41 shown in Figure 4, the output data of quantitative PCR is obtained. This can also be done by inputting the output data of the PCR detection obtained in step S03 shown in Figure 1 into a computer. The output data consists of numerical values ​​of the signal intensity representing the amount of amplified product or amplification event for each cycle number. The signal intensity may be, for example, RFU value (relative fluorescence unit). Here, the output data is discrete data separated by cycle number.

[0071] In step S42 shown in Figure 4, nonlinear regression is performed from the output data to the amplification curve. The amplification curve is continuous data. The amplification curve model used for this nonlinear regression may also be a Richard curve. Initial values ​​for the parameters that need to be substituted into the amplification curve model may be calculated in advance from the output data.

[0072] In step S43 shown in Figure 4, the second derivative of the amplification curve is calculated. Furthermore, in step S44, the number of cycles that yields the maximum value of the second derivative is set as the Cq value in the output data of the quantitative PCR. If the Cq value falls below a predetermined threshold for the number of cycles, this may be determined as the detection of the amplified product. The threshold may be determined experimentally in advance.

[0073] <Examples of mathematical analysis>

[0074] Figure 5 shows a flowchart illustrating the specific procedure for calculating the Cq value in steps S42 to S44 shown in Figure 4. In the flowchart, parameters or models are enclosed in rectangles. Arrows extending from a given parameter or model indicate the derivation of the next parameter or model. A detailed explanation will follow with reference to Figure 5.

[0075] As shown in Figure 5, quantitative PCR is first performed to obtain output data containing RFU values ​​for each cycle number. Next, linear interpolation is performed between adjacent cycles for the discrete RFU values ​​between cycles. This calculates the linear slope for each cycle number. The linear slope is obtained before and after each cycle. Using the obtained linear slope, discrete data of the slope for each cycle number is created. Linear interpolation is then performed on this discrete data. This gives the linear "slope of the slope" for each cycle number. Furthermore, the maximum value of the linear slope of the slope is calculated. This gives the base cycle (BC), the cycle number at which the slope of the slope is maximized.

[0076] As shown in Figure 5, the amplification curve is obtained by nonlinear regression to the Richard curve. Therefore, the maximum RFU value (Fmax), the number of cycles for half of the maximum RFU value (c), the background RFU value (Fb), the initial value of the slope of the amplification curve model (b), and the initial value of the Richard coefficient (d) are prepared in advance.

[0077] As shown in Figure 5, a maximum value prediction model is constructed in advance using cross-validation to obtain the maximum RFU value (Fmax). The maximum value prediction model is a model that predicts the maximum RFU value (Fmax) of the amplification curve. For construction, sample data of the slope before and after the number of cycles (BC) in which the slope of the amplification curve is maximized is used. This sample data is to be obtained in advance from multiple amplification curves. For example, a maximum value prediction model like the one below can be obtained in advance.

[0078]

number

[0079] As shown in Figure 5, the linear slopes before and after the base cycle (BC), obtained from the linear slopes for each cycle count, are applied to the maximum value prediction model to calculate the maximum RFU value (Fmax). In the formula, Slope1 is substituted with the linear slope before the base cycle (BC), and Slope2 is substituted with the linear slope after the base cycle (BC).

[0080] As shown in Figure 5, a half-valued prediction model is constructed in advance using cross-validation to obtain the half-valued cycle number (c) of the maximum RFU. The half-valued prediction model predicts the number of cycles that is half the maximum RFU value (Fmax) of the amplification curve. For construction, sample data of the number of cycles (BC) where the slope of the amplification curve is maximized is used. This sample data is to be obtained in advance from multiple amplification curves. For example, the following half-valued prediction model can be obtained in advance.

[0081]

number

[0082] As shown in Figure 5, the base cycle (BC) is applied to the half-valued prediction model to calculate the half-valued number of cycles (c) for the maximum RFU. In the formula, BC is substituted with the BC that corresponds to the maximum value of the slope of the linear slope described above.

[0083] As shown in Figure 5, the smallest RFU value among the RFU values ​​for each cycle count is defined as the background RFU value (Fb). Furthermore, the initial value (b) of the slope of the amplification curve obtained by nonlinear regression is set as appropriate. For example, 1.5. The initial value (d) of the Richard coefficient is set as appropriate. For example, 1.

[0084] Substitute the Fmax, c, Fb, b, and d described above into the Richard curve in the following equation. This will perform a nonlinear regression from the output data of quantitative PCR to the amplification curve.

[0085]

number

[0086] The second derivative F''x is calculated from the obtained amplification curve as follows.

number

[0087] In the obtained second derivative, x represents the number of cycles. Here, we calculate the maximum value of F''x in the range 0 ≤ x ≤ 40. The number of cycles that yields the obtained maximum value is set as the Cq value of the corresponding amplification curve. By using this Cq value, the detection of the amplification product can be determined as described above.

[0088] <Reagents and Kits>

[0089] In one embodiment, a reagent for monitoring viral sequences in virus-infected tissue is provided. The viral primer 26 shown in Figure 2 can be prepared based on information of known viral sequences. The reagent contains a viral primer 26 having a specific sequence. Its use is as described above. The reagent may be provided as a kit along with other substances necessary for PCR. The reagent may also be provided in a state where PCR can be initiated simply by mixing it with individualized primers 27. [Explanation of Symbols]

[0090] 10: Host, 11: Viral genome, 12: Host genome, 13: Insertion site, 14: Viral sequence, 15: Infection marker, 16: Neighboring sequence, 17: Non-neighboring sequence, 19: Diagnosis, 20: Blood, 22: Procedure, 23: Pathological specimen, 24: Monitoring, 25: Blood, 26: Viral primer, 27: Personalized primer, 30: Cell-free DNA

Claims

1. A method for monitoring viral sequences in virus-infected tissue, Using cell-free DNA from the host's blood as a template, Viral primers assigned to the viral genome sequence, Individualized primers assigned to the host genome sequence, Quantitative PCR was performed on the following groups. The amplification product, which includes the boundary between the viral genome and the host genome, is detected. Here, The individualized primer is assigned to a sequence near the site on the host genome sequence in which the virus is incorporated. The site on the genome sequence in which the virus is incorporated has been identified from a sample taken from the host before monitoring. method.

2. The aforementioned sample was obtained from tissue that had become diseased due to infection with the aforementioned virus. The method according to claim 1.

3. The sites identified from the diseased tissue are narrowed down based on the condition that they are also identified as sites where the virus is incorporated from cell-free DNA in blood collected from the host before monitoring. The method according to claim 2.

4. The aforementioned sample is cell-free DNA from blood. The fact that the aforementioned site originates from tissue that has become diseased due to infection with the virus is identified based on the fact that the host was diagnosed with the disease when blood was collected prior to monitoring. The method according to claim 1.

5. The aforementioned viral primers are specific to the species or subtype of the virus identified by genotyping of free viruses collected from the vicinity of the virus-infected tissue prior to monitoring. The method according to claim 1.

6. The free virus is collected from the host before the sample is collected from the host. The method according to claim 5.

7. The disease occurring in tissues infected with the aforementioned virus is cancer. The method according to claim 1.

8. The aforementioned virus is a DNA virus. The method according to claim 1.

9. The disease occurring in tissues infected with the aforementioned virus is cervical cancer. The aforementioned virus is human papillomavirus (HPV), The aforementioned tissue is the tissue of the cervix. The method according to claim 1.

10. The vicinity of the site where the virus is incorporated refers to the range that can be amplified by PCR, starting from the site where the virus is incorporated. The method according to claim 1.

11. The presence or absence of detection of the amplification product, or the amount of the amplification product detected, is provided as data for diagnosing a disease occurring in tissue infected with the virus. The method according to any one of claims 1 to 9.

12. A reagent for monitoring viral sequences in virus-infected tissue, It contains viral primers assigned to the viral genome sequence, In that usage, Using cell-free DNA from the host's blood as a template, The aforementioned virus primer, Individualized primers assigned to the host genome sequence, PCR was performed in the group. The amplification product, which includes the boundary between the viral genome and the host genome, is detected. Here, The individualized primer is assigned to a sequence near the site on the host genome sequence in which the virus is incorporated. The site in which the virus is incorporated has been identified from a sample taken from the host before monitoring. reagent.

13. In that usage, When the sample was collected from the host before monitoring, the host was suffering from a disease caused by infection with the virus. If the detected amount of the amplification product exceeds the standard value, the host is diagnosed with a recurrence of the disease. The reagent according to claim 12.

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