CN116656794A - Digital PCR detection kit for tuberculosis detection based on four-gene scoring method - Google Patents

Digital PCR detection kit for tuberculosis detection based on four-gene scoring method Download PDF

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CN116656794A
CN116656794A CN202310519891.0A CN202310519891A CN116656794A CN 116656794 A CN116656794 A CN 116656794A CN 202310519891 A CN202310519891 A CN 202310519891A CN 116656794 A CN116656794 A CN 116656794A
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tuberculosis
gene
seq
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赵雁林
邱倩
欧喜超
赵冰
夏江
高学娟
余皓
金雨璐
陈耀凯
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Pilot Gene Technologies Hangzhou Co ltd
Chinese Center for Disease Control and Prevention
Chongqing Public Health Medical Center
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Pilot Gene Technologies Hangzhou Co ltd
Chinese Center for Disease Control and Prevention
Chongqing Public Health Medical Center
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Abstract

The application provides a digital PCR detection kit for detecting tuberculosis based on a four-gene scoring method, which utilizes a digital PCR amplification technology to detect the transcription level of four tuberculosis infection host marker genes in different channels of the same amplification system simultaneously, and does not need normalization of housekeeping genes. In order to avoid the difference caused by different template total amounts during amplification, the method adopts the detection values of IFIT3 genes with moderate transcription levels in four host genes as denominators to carry out standardized treatment on gene detection values of other channels, calculates four-gene scoring by utilizing the change relation of the transcription levels of four tuberculosis infection host marker genes, judges tuberculosis by the four-gene scoring, can effectively identify tuberculosis latency and active tuberculosis, and has the effects of higher specificity and more accurate results. In addition, the scheme designs a primer probe sequence aiming at four tuberculosis infection host marker genes, and can more accurately detect the corresponding gene transcription level.

Description

Digital PCR detection kit for tuberculosis detection based on four-gene scoring method
Technical Field
The application relates to the field of genes, in particular to a digital PCR detection kit for tuberculosis detection based on a four-gene scoring method.
Background
Tuberculosis is a chronic infectious disease caused by mycobacterium tuberculosis, can invade many viscera, and is most common in pulmonary tuberculosis infection. The part of adult infected with mycobacterium tuberculosis presents with bacteria survival state, and does not develop into tuberculosis, and the situation is called tuberculosis latent infected person, and about 5-10% of tuberculosis latent infected person can develop into active tuberculosis. Active tuberculosis refers to that tuberculosis focus is in active stage, and there is often patch-like shadow or tuberculosis cavity on chest film, or focus is disseminated, which indicates that mycobacterium tuberculosis is actively propagated, virulence is strong, and a large number of tuberculosis latent infected people without clinical symptoms are important sources of active tuberculosis. Furthermore, since the prophylactic treatment regimen for latent tuberculosis infection is significantly different from the anti-tuberculosis treatment regimen for active tuberculosis. Therefore, it is important how to achieve early differential diagnosis of a latent mycobacterium tuberculosis infected person from an active tuberculosis patient.
In the prior art, the detection of tuberculosis mainly comprises the following means: x-ray imaging examinations, however, these X-ray imaging examinations are only suitable for the case where tuberculosis lesions are more evident, and can not detect patients with tuberculosis latency, and can not distinguish tuberculosis and other lesions well. 2. Tuberculin experiments, this approach is susceptible to interference from other pathogens and does not distinguish tuberculosis infection latency from active tuberculosis well. 3. The molecular biological detection technology based on nucleic acid amplification, but the mode is influenced by primer probe selection and amplification conditions, and at present, no good scheme can effectively distinguish the situations of latent tuberculosis and active tuberculosis.
Disclosure of Invention
The application aims to provide a digital PCR detection kit for detecting tuberculosis based on a four-gene scoring method, which can effectively identify latent tuberculosis and active tuberculosis by detecting the transcription level of four tuberculosis infection host marker genes of IFIT1, IFIT3, OAS1 and CXCL10, calculating the score of the transcription level of the four tuberculosis infection host marker genes by the four-gene scoring method, and carrying out effective tuberculosis detection based on the obtained score.
The scheme selects the transcription level of four tuberculosis infection host marker genes, namely IFIT1, IFIT3, OAS1 and CXCL10, to realize the accurate detection of the tuberculosis infection. The four tuberculosis infection host marker genes can change to a certain extent when the host is infected with mycobacterium tuberculosis, and IFIT1 and IFIT3 are interferon stimulating genes, have antiviral and pro-inflammatory functions, and have research marks: IFIT1 is a negative regulator of the pro-inflammatory cytokine gene TNF, while IFIT1 has negative feedback regulation in the IFNB1 gene induction and antiviral IFN gene programs. The IFIT1 and IFIT3 proteins have been widely studied as important viral limiting factors, having a range of established antiviral functions. OAS1 is a regulator of interferon-induced pathophysiology of high inflammatory monocytes and B lymphocytes. IP-10 is a protein encoded by CXCL10 gene and has higher expression level after stimulation by Mycobacterium tuberculosis antigen.
In addition, the detection system developed by the scheme uses different channels of the same amplification system to detect four genes simultaneously, that is, four tuberculosis infection host marker genes of one sample are amplified in the same amplification tube, so that the advantage is that the sample loading amount can be ensured to be completely consistent without normalization of the housekeeping genes. And because the content difference of mononuclear cells of different patients is large, the difference of the collection amount of mononuclear cells is also large, so the total RNA amount of different samples is different, even if templates with the same volume are added during detection, the difference of detection values is large, in order to avoid the difference caused by the difference of the total amounts of the templates during amplification, the method selects IFIT3 detection values with moderate transcription levels in four tuberculosis infection host marker genes as denominators, the detection values of other tuberculosis infection host marker genes are used as molecules for carrying out detection value standardization treatment, the detection values of the transcription levels of the four tuberculosis infection host marker genes are utilized for scoring, and whether the patient is infected by the tuberculosis mycobacterium or not and the type of infection belongs to tuberculosis latency or activity stage are judged based on the obtained scores.
In a first aspect, the present disclosure provides an application of a four-gene scoring-based tuberculosis detection, wherein the detected tuberculosis infection host marker genes are IFIT1, IFIT3, OAS1, CXCL10, comprising: obtaining transcription levels of tuberculosis infection host marker genes IFIT1, IFIT3, OAS1 and CXCL10, and calculating according to the following formula to obtain four-gene scoring:
four-gene scoring = Bf1/bf3+b0/bf3+bc/Bf3;
wherein Bf1 is the transcript level of IFIT1, bf3 is the transcript level of IFIT3, BO is the transcript level of OAS1, bc is the transcript level of CXCL10, if the four-gene score is greater than a first threshold, the infection of the patient by mycobacterium tuberculosis is judged, tuberculosis latency is between the first threshold and a second threshold, and active tuberculosis is greater than the second threshold.
In some embodiments, the first threshold is 2.2 and the second threshold is 2.8. In other words, if the four-gene score is greater than 2.8, it is judged as active tuberculosis; if the four-gene score is between 2.2 and 2.8, judging that the tuberculosis is latent, and if the four-gene score is less than 2.2, judging that the tuberculosis is not infected by the mycobacterium tuberculosis.
In some embodiments, detection of transcript levels of marker genes of four tuberculosis-infected hosts is performed by a digital PCR method. The central idea of digital PCR is to detect fluorescent signals of all reaction units after partition amplification, directly calculate the concentration or copy number of target molecules by using digital model correction, and realize quantitative analysis without comparing standard samples and standard curves and depending on CT values.
In some embodiments, four tuberculosis infection host marker genes are detected in different channels of the same amplification system, and the detected value of the corresponding channel is obtained as the transcription level of the tuberculosis infection host marker genes.
In some implementations, the specific amplified tuberculosis infection host marker genes IFIT1, IFIT3, OAS1, CXCL10 and the exogenous internal reference primer probe are mixed to obtain a primer probe mixed solution, the primer probe mixed solution is mixed to prepare a ddPCR reaction system, and RNA extracted from a blood sample to be detected and the ddPCR reaction system are mixed and then amplified and detected under a set amplification program to obtain the detection of the transcription level of the tuberculosis infection host marker genes.
In some embodiments, the primer sequences for specifically amplifying the tuberculosis infection host marker gene IFIT1 are shown in SEQ ID NO. 1 and SEQ ID NO. 2, the probe sequences are shown in SEQ ID NO. 3, and the corresponding marker sequences are shown in SEQ ID NO. 0:17. In some embodiments, the modified fluorophores that specifically detect the probe sequence of tuberculosis infection host marker gene IFIT1 are FAM and MGB.
The primer sequences of the specific amplified tuberculosis infection host marker gene IFIT3 are shown as SEQ ID NO. 4 and SEQ ID NO. 5, the probe sequence is shown as SEQ ID NO. 6, and the corresponding marker sequence is shown as SEQ ID NO. 0:18.
In some embodiments, the modified fluorophores that specifically detect the probe sequence of the tuberculosis infection host marker gene IFIT3 are ROX and MGB.
The primer sequences of the specific amplified tuberculosis infection host marker gene OAS1 are shown as SEQ ID NO. 7 and SEQ ID NO. 8, the probe sequence is shown as SEQ ID NO. 9, and the corresponding marker sequence is shown as SEQ ID NO. 0:19. In some embodiments, the modified fluorophores that specifically detect the probe sequence of tuberculosis infection host marker gene OAS1 are VIX and MGB.
The primer sequences for specifically detecting the tuberculosis infection host marker gene CXCL10 are shown as SEQ ID NO. 10 and SEQ ID NO. 11, the probe sequences are shown as SEQ ID NO. 12, and the corresponding marker sequences are shown as SEQ ID NO. 0 and 20. In some embodiments, the modified fluorescent genes of the probe sequences that specifically detect the tuberculosis infection host marker gene CXCL10 are CY5 and MGB.
The scheme also randomly generates random primers shown as SEQ ID NO. 13-14 and a probe shown as SEQ ID NO. 15 as exogenous internal references, and the corresponding marker sequence is shown as SEQ ID NO. 0-16.
The specific sequences are shown in table one below:
in a second aspect, the present disclosure provides a digital PCR detection kit for tuberculosis detection based on a four-gene scoring method, comprising:
the primer sequences of the specific amplified tuberculosis infection host marker gene IFIT1 are shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the probe sequence is shown as SEQ ID NO. 3;
the primer sequences of the specific amplified tuberculosis infection host marker gene IFIT3 are shown as SEQ ID NO. 4 and SEQ ID NO. 5, and the probe sequence is shown as SEQ ID NO. 6;
the primer sequences of the specific amplified tuberculosis infection host marker gene OAS1 are shown as SEQ ID NO. 7 and SEQ ID NO. 8, and the probe sequences are shown as SEQ ID NO. 9;
the primer sequences for specifically detecting the tuberculosis infection host marker gene CXCL10 are shown as SEQ ID NO. 10 and SEQ ID NO. 11, and the probe sequences are shown as SEQ ID NO. 12.
In some embodiments, the digital PCR detection kit further comprises random primers as shown in SEQ ID NOS 13-14 and a probe as shown in SEQ ID NO 15 as exogenous internal references.
In some embodiments, the digital PCR detection kit further comprises: water, ddPCR Mix, a marker sequence shown as SEQ ID No. 0:16.
In some embodiments, the digital PCR detection kit is used for digital PCR detection, detecting in different channels of the same amplification tube, obtaining detection values of different channels as transcription levels of corresponding tuberculosis infection host marker genes, and calculating based on the following formula to obtain a four-gene score:
four-gene scoring = Bf1/bf3+b0/bf3+bc/Bf3;
wherein Bf1 is the transcript level of IFIT1, bf3 is the transcript level of IFIT3, BO is the transcript level of OAS1, bc is the transcript level of CXCL10, and Mycobacterium tuberculosis infection is judged if the four-gene score is greater than a set threshold.
In some embodiments, the amplification procedure is: 95 ℃ for 5min [ 15s at 95 ℃, 30s at 60) ].
In some embodiments, the test is performed on a blood sample of the patient to be tested.
In a third aspect, the present solution provides an application of a digital PCR detection kit for tuberculosis detection based on a four-gene scoring method, for tuberculosis detection for non-diagnostic purposes, and for distinguishing between latent tuberculosis or active tuberculosis.
Compared with the prior art, the technical scheme has the following characteristics and beneficial effects:
the scheme utilizes the digital PCR amplification technology to detect the transcription level of four tuberculosis infection host marker genes in different channels of the same amplification tube simultaneously, and normalization of housekeeping genes is not needed. In order to avoid the difference caused by different template total amounts during amplification, the method adopts the detection values of IFIT3 genes with moderate transcription levels in four host genes as denominators to carry out standardized treatment on gene detection values of other channels, calculates four-gene scoring by utilizing the change relation of the transcription levels of four tuberculosis infection host marker genes, judges tuberculosis infection by the four-gene scoring, can effectively identify tuberculosis latency and active tuberculosis, and has the effects of higher specificity and more accurate results. In addition, the scheme designs a primer probe sequence aiming at four tuberculosis infection host marker genes, and can more accurately detect the corresponding gene transcription level.
Drawings
Fig. 1 is a scatter plot of test results for 100 patient clinical blood samples.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which are derived by a person skilled in the art based on the embodiments of the application, fall within the scope of protection of the application.
In order to verify the feasibility of the scheme, the inventor constructs a digital PCR detection system and performs the following experimental verification:
construction of a digital PCR detection system:
mixing to obtain a primer probe mixed solution, wherein the mixed system of the primer probe mixed solution is shown in the following table II:
mixed liquid system of surface two primer probe
Mixing to obtain a ddPCR reaction system, wherein the ddPCR reaction system is shown in the following Table III:
table three ddPCR reaction system
Reagent(s) Volume (mu L) Final concentration
Water and its preparation method 4.735
Primer probe mixed liquid 1.265
5XddPCR Mix 3 1X
IC-S 1
DNA 5
Totalizing 15
The DNA is the DNA of the sample to be tested.
The amplification procedure was as follows:
95℃5min【95℃15s，60℃30s】*40。
and (3) verification:
8 normal blood samples are selected for detection, RNA is extracted by a column method, and common infectious microbe DNA samples are selected: and (5) outsourcing the inactivated bacteria, and extracting genome DNA by a magnetic bead method. The test results are shown in Table IV below:
blood sample detection for four 8 normal people
Therefore, the specificity of the system for detecting various common bacteria and fungi is normal, and blood samples of 8 normal people are scored between 1.79 and 2.2 by the detection of the kit, so that the repeatability is good.
Embodiment one: blank detection limit (LoB) value:
extracting 20 parts of deionized water every day as a template, respectively using the ddPCR reaction systems on different amplification instruments for three continuous days, analyzing whether the detection result is subjected to normal distribution by using spss23.0 software S-W except that all detection values are 0, and analyzing LoB values by using a parameter method if the detection result is subjected to normal distribution; if normal distribution is not obeyed, the LoB value is analyzed by a nonparametric method. The results are shown in Table five below:
five blank detection limit value
IFIT1 IFIT3 OAS1 CXCL10
First day 0.089 0 0.18 0.09
The next day 0 0.09 0 0.11
Third day 0.09 0 0.09 0.18
The detection target takes a maximum value of LoB from 3 calculations, i.e. IFIT1 and IFIT3 of 0.09 copies/. Mu.l, and OAS1 and CXCL10 of 0.18 copies/. Mu.l.
Example 2 blank detection Limit (LoD) value
1.1 minimum detection limit (LoD)
If LoB =0, a probability unit scheme is adopted; if LoB is not equal to 0, the classical scheme is adopted. The maximum LOD in 3 batches of reagents was formulated as the final reported value.
1.1.1 probability Unit scheme
The DNA of known concentrations of different target genes was incorporated as positive samples using ultrapure water as a substrate. Then, ultrapure water was used for gradient dilution.
The same set of instrument, including a droplet generator, a PCR amplification instrument and a chip reader, is used for repeatedly detecting different concentration gradients of each target gene specimen 30 times respectively by using 3 batch reagents.
And respectively calculating the positive detection rate of each concentration gradient of each target gene specimen, and selecting the lowest concentration with the positive detection rate more than or equal to 95% as the LoD value of each target gene.
1.1.2 classical scheme
The DNA of known concentrations of different target genes was incorporated as positive samples using ultrapure water as a substrate. Then, the samples were diluted to five concentrations of 1X LoB, 2X LoB, 3X LoB, 4X LoB and 5X LoB, respectively, using the corresponding ultrapure water, and used as low-value samples.
The same set of instrument, including a droplet generator, a PCR amplification instrument and a chip reader, is used for repeatedly detecting five low-value samples of each target gene at least 12 times respectively by using 3 batch reagents, and the total of at least 60 low-value sample measurement results are obtained.
The method for calculating the LoD value of each target gene comprises the following steps:
1) The data distribution of at least 60 low value specimen measurements per lot reagent is analyzed.
2) If the data are normally distributed, the analysis is carried out by adopting a parameter statistical method.
3) If the data is distributed in a biased state, adopting a non-parameter statistical method to analyze.
The results are shown in Table six below:
six blank detection limit values
The detection limit was LoD, IFIT1, IFIT3 and OAS1 at 0.18 copies/. Mu.l and CXCL10 at 0.36 copies/. Mu.l.
Embodiment three clinical thresholding:
the results of volunteer tests were determined by testing 150 cases of patients with positive and tuberculous onset of the gamma interferon release test, 150 cases of non-onset patients with positive gamma interferon release test, and 150 cases of negative gamma interferon release test. The four gene scoring detection threshold lines of normal people and tuberculosis latency are marked as 2.2, and the detection threshold lines of tuberculosis latency and active tuberculosis are marked as 2.8.
As shown in table seven below:
table seven score
0-2.20 2.20-2.80 ≥2.80
Four-gene scoring value Normal person Latent tuberculosis Active tuberculosis
Therefore, the scheme selects 2.2 and 2.8 as the four-gene scoring threshold, if the threshold is more than 2.2, the mycobacterium tuberculosis infection is considered to exist, the tuberculosis latency is considered to be in the interval of 2.2-2.8, and the active tuberculosis is considered to be in the interval of more than 2.8.
Example four true sample verification:
on the basis of the detection threshold line, 100 patient blood samples with unknown clinical information of the contact history of the tuberculosis patient are detected, the sample information of the patient is recorded in detail in the detection process, the clinical information of the patient is checked according to the detection result, and the clinical information of the patient is checked according to the detection result, so that the detection result is made into a scatter diagram record as shown in figure 1.
The clinical information is checked to obtain: six of four-gene scoring values greater than 2.8 are tuberculosis patients undergoing treatment; 51 patients with positive gamma interferon release test detection at four gene scoring values between 2.2 and 2.8; the other 43 cases were patients whose gamma interferon release test was negative.
The present application is not limited to the above-mentioned preferred embodiments, and any person who can obtain other various products under the teaching of the present application can make any changes in shape or structure, and all the technical solutions that are the same or similar to the present application fall within the scope of the present application.

Claims (10)

1. An application of tuberculosis detection based on a four-gene scoring method, wherein the detected tuberculosis infection host marker genes are IFIT1, IFIT3, OAS1 and CXCL10, and the method comprises the following steps: obtaining transcription levels of tuberculosis infection host marker genes IFIT1, IFIT3, OAS1 and CXCL10, and calculating according to the following formula to obtain four-gene scoring:
four-gene scoring = Bf1/bf3+b0/bf3+bc/Bf3;
wherein Bf1 is the transcript level of IFIT1, bf3 is the transcript level of IFIT3, BO is the transcript level of OAS1, bc is the transcript level of CXCL10, if the four-gene score is greater than a first threshold, the patient is judged to have a Mycobacterium tuberculosis infection, tuberculosis latency is between the first threshold and a second threshold, and the patient is greater than the second threshold and is an active tuberculosis patient.
2. The use of four-gene scoring-based tuberculosis detection as described in claim 1, wherein the detection of transcript levels of marker genes of four tuberculosis-infected hosts is performed based on a digital PCR method.
3. The use of four-gene scoring-based tuberculosis detection as described in claim 2, wherein four tuberculosis infection host marker genes are detected in different channels of the same amplification system, and the detected value of the corresponding channel is obtained as the transcription level of the tuberculosis infection host marker genes.
4. The use of four-gene scoring-based tuberculosis detection as described in claim 1, wherein the first threshold is 2.2 and the second threshold is 2.8.
5. A digital PCR detection kit for tuberculosis detection based on a four-gene scoring method, comprising:
the primer sequences of the specific amplified tuberculosis infection host marker gene IFIT1 are shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the probe sequence is shown as SEQ ID NO. 3;
the primer sequences of the specific amplified tuberculosis infection host marker gene IFIT3 are shown as SEQ ID NO. 4 and SEQ ID NO. 5, and the probe sequence is shown as SEQ ID NO. 6;
the primer sequences of the specific amplified tuberculosis infection host marker gene OAS1 are shown as SEQ ID NO. 7 and SEQ ID NO. 8, and the probe sequences are shown as SEQ ID NO. 9;
the primer sequences for specifically detecting the tuberculosis infection host marker gene CXCL10 are shown as SEQ ID NO. 10 and SEQ ID NO. 11, and the probe sequences are shown as SEQ ID NO. 12.
6. The digital PCR detection kit for tuberculosis detection based on the four-gene scoring method as described in claim 5, wherein the kit comprises random primers shown as SEQ ID NO. 13-14 and a probe shown as SEQ ID NO. 15 as exogenous internal reference.
7. The digital PCR detection kit for tuberculosis detection based on the four-gene scoring method as described in claim 5, wherein the detection is performed in different channels of the same amplification system for digital PCR detection, and the detected values of the different channels are obtained as the transcription level of the corresponding tuberculosis infection host marker gene, based on the four-gene scoring calculated according to the following formula:
four-gene scoring = Bf1/bf3+b0/bf3+bc/Bf3;
wherein Bf1 is the transcript level of IFIT1, bf3 is the transcript level of IFIT3, BO is the transcript level of OAS1, bc is the transcript level of CXCL10, if the four-gene score is greater than a first threshold, the patient is judged to have a Mycobacterium tuberculosis infection, tuberculosis latency is between the first threshold and a second threshold, and the patient is greater than the second threshold and is an active tuberculosis patient.
8. The digital PCR detection kit for four-gene scoring-based tuberculosis detection of claim 7, wherein the first threshold is 2.2 and the second threshold is 2.8.
9. Use of a digital PCR detection kit for tuberculosis detection based on the four-gene scoring method according to any one of claims 5 to 8, characterized by tuberculosis latency and active tuberculosis detection for non-diagnostic purposes.
10. The use of a digital PCR detection kit for tuberculosis detection based on the four-gene scoring method as in claim 9, wherein the primer probe for specifically amplifying the tuberculosis infection host marker genes, i.e., IFIT1, IFIT3, OAS1, CXCL10 and exogenous internal reference, is mixed to obtain a primer probe mixture, the primer probe mixture is mixed to prepare a ddPCR reaction system, and RNA extracted from a blood sample to be detected and the ddPCR reaction system are mixed and then amplified and detected under a set amplification program to obtain the detection of the transcription level of the tuberculosis infection host marker genes.
CN202310519891.0A 2023-05-09 2023-05-09 Digital PCR detection kit for tuberculosis detection based on four-gene scoring method Pending CN116656794A (en)

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