A polynucleotide, a kit, an isothermal detection method and a method for predicting preterm birth

Abstract

A polynucleotide, a kit, an isothermal detection method and a method for predicting premature birth, the method for predicting premature birth comprising: analyzing the expression profile of any one or at least two gene sets in a maternal sample, and predicting whether the subject will have premature birth according to the expression profile. The present application detects the expression amount of the marker gene in the serum of pregnant women for the first time, which is used for the prediction of premature birth risk of pregnant women without clinical symptoms. The main advantage is serological detection, no need to make an appointment in advance like ultrasound, non-invasive detection, no need for vaginal or cervical sampling, and good detection process experience.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medicine, specifically to a polynucleotide, a reagent kit, an isothermal detection method, and a method for predicting preterm birth. Background Technology

[0002] Preterm birth is defined as delivery before 37 weeks of gestation. Globally, the incidence of preterm birth is approximately 10%, resulting in about 15 million preterm births annually. In China, preterm birth accounts for about 7.3% of all newborns. Preterm birth can lead to various neonatal complications and is one of the most common causes of neonatal death, imposing heavy family expenses and a significant burden on social healthcare. However, the etiology and pathogenesis of preterm birth are not yet fully understood, and there is a lack of targeted and effective prevention and treatment measures, as well as effective and safe screening methods in early pregnancy.

[0003] Traditional methods for screening for preterm birth include cervical length, fetal fibronectin, interleukin, and insulin-like growth factor binding protein 1 (IGF-1). At 24 weeks of gestation, a cervical length of less than 25 mm has a sensitivity of approximately 37.3% and a specificity of 92.2% in predicting preterm birth risk. Fetal fibronectin is a protein secreted by fetal amniotic cells and trophoblast cells. It can be detected in vaginal secretions before 22 weeks of gestation, and its presence between 24 and 34 weeks indicates a risk of preterm birth. Its sensitivity and specificity are higher when the mother has clinical manifestations related to preterm birth, but its predictive effect is poor for asymptomatic spontaneous preterm birth. For asymptomatic preterm births before 34 weeks of gestation, the area under the ROC curve is 0.61. Insulin-like growth factor binding protein 1 (IGF-1) plays a crucial role in placental growth and development. Abnormal development can lead to the secretion of this protein into cervical mucus, potentially enabling early diagnosis. Detection of IGF-1 in cervical mucus between 24 and 34 weeks of gestation can predict preterm labor. For asymptomatic preterm women, the predictive sensitivity is approximately 14–47%, and the specificity is approximately 76–93%. For preterm women with clinical manifestations, the predictive sensitivity is approximately 60–68%, and the specificity is approximately 77–81%. Inflammatory factors are associated with prostaglandin synthesis. The presence of interleukin-6 (IL-6) in cervical mucus is associated with preterm labor. In individuals with preterm labor manifestations, the sensitivity of IL-6 is approximately 69.4%, and the specificity is approximately 68.2%.

[0004] Traditional methods for predicting preterm birth have unsatisfactory sensitivity and specificity, making them difficult to meet clinical needs. These methods typically predict preterm birth well in individuals with clinical symptoms, but their predictive efficacy is poor in those without symptoms. Moreover, most of these tests require vaginal or cervical sampling, which is not user-friendly for pregnant women. Summary of the Invention

[0005] According to a first aspect, in one embodiment, a polynucleotide is provided, the polynucleotide comprising a first anchoring sequence and a first guide sequence connected in tandem, the first anchoring sequence being bindable to a nuclease, the first guide sequence being specific to a target nucleic acid molecule and capable of specifically binding to the target nucleic acid molecule, the first anchoring sequence binding to the nuclease, and the first guide sequence binding to the target nucleic acid molecule causing the target nucleic acid molecule on the target nucleic acid molecule to be cleaved.

[0006] According to a second aspect, in one embodiment, a kit is provided comprising the polynucleotide described in the first aspect.

[0007] According to a third aspect, in one embodiment, the use of at least one of the genes shown in Table 1 as a biomarker in the preparation and / or screening of gestational age prediction reagents is provided.

[0008] According to the fourth aspect, in one embodiment, a method for predicting preterm birth is provided, comprising: analyzing the expression profile of any one or at least two genes in a maternal sample, and predicting whether the subject will be premature based on the expression profile.

[0009] According to the fifth aspect, in one embodiment, an isothermal enzyme digestion detection method is provided, comprising: performing an enzyme digestion reaction on a template to be tested under isothermal conditions, and collecting fluorescence values ​​during and / or at the end of the enzyme digestion reaction.

[0010] According to a sixth aspect, in one embodiment, a system for predicting preterm birth is provided, comprising: a preterm birth analysis device for analyzing the expression profile of any one or at least two genes in a maternal sample, and predicting whether a subject will be born prematurely based on the expression profile.

[0011] According to a seventh aspect, in one embodiment, an apparatus is provided, comprising:

[0012] Memory, used to store programs;

[0013] A processor for implementing the method described in the fourth aspect by executing a program stored in the memory.

[0014] According to the eighth aspect, in one embodiment, a computer-readable storage medium is provided, the medium storing a program that can be executed by a processor to implement the method as described in the fourth aspect.

[0015] Based on the polynucleotide, reagent kit, isothermal detection method, and method for predicting preterm birth described in the above embodiments, this invention is the first to detect the expression levels of marker genes in the serum of pregnant women for predicting the risk of preterm birth in asymptomatic pregnant women. Its main advantages lie in serological testing, which does not require prior appointments like ultrasound, is non-invasive, does not require vaginal or cervical sampling, and offers a superior testing experience. The method of this invention is simple and low-cost. It exhibits high sensitivity and specificity, allows for repeated monitoring, and has better clinical application prospects compared to traditional methods. Attached Figure Description

[0016] Figure 1 A distribution of preterm delivery times for maternal samples used in transcriptome sequencing;

[0017] Figure 2 A distribution of full-term delivery times for maternal samples used in transcriptome sequencing;

[0018] Figure 3 A distribution of preterm birth sample collection time for maternal samples used in transcriptome sequencing;

[0019] Figure 4 A distribution of full-term sample collection time for maternal samples used in transcriptome sequencing;

[0020] Figure 5 A statistical graph showing the expression levels of LMCD1, SNAPC1, and RETSAT genes;

[0021] Figure 6 ROC curve of the training set during modeling;

[0022] Figure 7 ROC curve for the test set during modeling;

[0023] Figure 8 A gestational week distribution of full-term deliveries during clinical sample validation;

[0024] Figure 9 A gestational week distribution of preterm deliveries during clinical sample validation;

[0025] Figure 10 A gestational week distribution map for full-term samples taken during clinical sample validation;

[0026] Figure 11 A gestational week distribution map of preterm birth samples during clinical sample validation;

[0027] Figure 12 ROC curve for the training set during clinical sample validation;

[0028] Figure 13 ROC curve for the test set during clinical sample validation;

[0029] Figure 14 This is a schematic diagram of the enzyme digestion reaction principle. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings. Similar elements in different embodiments are referred to by associated similar element reference numerals. In the following embodiments, many details are described to facilitate a better understanding of this application. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other elements, materials, or methods. In some cases, certain operations related to this application are not shown or described in the specification. This is to avoid obscuring the core parts of this application with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; they can fully understand the related operations based on the description in the specification and general technical knowledge in the art.

[0031] Furthermore, the features, operations, or characteristics described in the specification can be combined in any suitable manner to form various embodiments. At the same time, the steps or actions in the method description can be rearranged or adjusted in a manner obvious to those skilled in the art. Therefore, the various orders in the specification and drawings are only for the clear description of a particular embodiment and do not imply a necessary order, unless otherwise stated that a particular order must be followed.

[0032] The serial numbers assigned to components in this document, such as "first" and "second," are used only to distinguish the described objects and have no sequential or technical meaning. Unless otherwise specified, the terms "connection" and "linkage" used in this application include both direct and indirect connections (linkages).

[0033] definition

[0034] As used herein, the term "cell-free RNA" or "cfRNA" refers to RNA, particularly mRNA, expressed by cells of the mother, fetus, and / or placenta and recoverable from the non-cellular portion of maternal blood, and comprising fragments of full-length RNA transcripts. In some embodiments, "cfRNA" does not contain rRNA. In some embodiments, "cfRNA" does not contain miRNA. In some embodiments, "cfRNA" refers to mRNA. cfRNA can also be recovered from maternal urine.

[0035] As used herein, the term “expression level” is equivalent to “expression profile,” and “expression profile” refers to the expression level of one or more gene products obtained from a maternal sample. The gene product can be cfRNA or protein. For gene products recovered from maternal plasma, the expression level can be expressed as the number of transcripts of a specific RNA per mL of maternal plasma, the mass of a specific polypeptide per mL of maternal plasma, the number of transcripts calculated by RNA-Seq, or any other suitable unit. Similar units can be used for gene products obtained from other maternal samples (e.g., urine). The expression of a gene product can be determined using any suitable method (e.g., as described below). Measurements are typically normalized to account for variations in sample quantity and quality, reverse transcription efficiency, etc. When an expression profile reflects the expression of multiple different gene products (e.g., different cfRNA transcripts), different weights can be assigned to the gene products when generating or comparing expression profiles or reference profiles. For example, when comparing expression profiles of cfRNA1 and cfRNA2 in samples from pregnant women with a reference profile (discussed below), a two-fold difference in cfRNA1 values ​​can be given greater weight than a two-fold difference in cfRNA2 values ​​when determining the degree of similarity or difference between the expression profiles and the reference profile. Expression profiles from maternal (e.g., patient) samples are sometimes referred to as “maternal expression profiles,” while maternal expression profiles in samples collected at a specified time can be referred to as “[time] maternal expression profiles,” such as “24-week maternal expression profiles.”

[0036] As used herein, a “reference profile” is an expression profile derived from a reference population. For illustration, an example of a reference population is pregnant women, women who deliver at term, or women who deliver prematurely. In some implementations, the reference population is a subgroup of pregnant women characterized by maternal age (e.g., women aged 20–25 years who deliver at term), race, or ethnicity (e.g., European Chinese women, American Chinese women, African Chinese women, etc. who deliver at term). The reference profile is generated by combining the expression profiles of a statistically significant number of women in the population. For a given gene product, the reference profile may reflect the average transcription level in the population, the median transcription level in the population, or can be determined using any of the many methods known in the fields of epidemiology and medicine. The reference population will typically include at least 10 subjects (e.g., 10–200 subjects), sometimes 50 or more subjects, and sometimes 1000 or more subjects.

[0037] As used herein, the term “spectrum set” refers to a collection of gene products measured in a particular assay. For example, in an assay for three (3) different cfRNAs (“RNA AF”), these three cfRNAs would be a spectrum set. Similarly, in an assay for three (3) different proteins from maternal plasma or urine, these three proteins would be a spectrum set. As another example, in an assay that collects expression data of transcripts of a large number of genes (e.g., the entire transcriptome or a large number of placental gene transcripts), a subset used to estimate gestational age or time of delivery or to assess the risk of preterm birth can be called a spectrum set. Measurements of RNA or proteins not included in the set can be used as controls to normalize measurements within or between samples, or for similar purposes. In some embodiments, the spectrum set may comprise a collection of gene products that includes both cfRNAs and proteins. The spectrum set is sometimes referred to as a “set”.

[0038] As used in this article, the terms "preterm birth," "full-term pregnancy," "full-term delivery," and "normal full-term pregnancy" have their normal meanings. Full-term refers to delivery after the fetus has reached 37 weeks of gestational age, while preterm birth refers to delivery before the fetus reaches 37 weeks of gestational age. In some cases, preterm birth refers to delivery between 16 and 35 weeks of gestational age or between 24 and 30 weeks of gestational age.

[0039] As used herein, "maternal sample" refers to a sample of bodily fluids obtained from a pregnant woman. Bodily fluids are typically serum, plasma, or urine, and are usually serum. In some embodiments, samples of different bodily fluids may be used, such as saliva, cerebrospinal fluid, pleural effusion, etc. Maternal samples can be obtained at multiple different times during pregnancy and stored (e.g., frozen) until testing. It should be understood that the date of collection of the maternal sample is an indispensable characteristic of the sample.

[0040] As used in this article, "time of delivery" refers to the number of weeks from the start of pregnancy to the date of delivery or the expected date of delivery. The time of delivery is calculated by subtracting the start of pregnancy from the time of delivery. The start of pregnancy is usually estimated, for example, from the first day of the last menstrual period before pregnancy. It can also be estimated through ultrasound examination, or by other methods such as the onset of morning sickness, the onset of fetal movement, the height of the fundus, and mid-pregnancy ultrasound. In this article, the sampling time is calculated by subtracting the start of pregnancy from the time of sampling, i.e., gestational age.

[0041] As used herein, the terms "protein" and "peptide" are used interchangeably. Mentioning a protein obtained from a maternal sample does not necessarily mean that the protein is a full-length gene expression product. Partial, fragment, and cleavage products can be detected and identified according to the present invention.

[0042] Unless otherwise specified, "serum" in this article refers to the colloidal liquid of plasma after fibrinogen has been removed. Plasma refers to the extracellular matrix of blood.

[0043] In this article, the Gene ID of the "LMCD1 gene" is 29995, and the transcript is NM_014583.4.

[0044] In this article, the gene ID of the "SNAPC1 gene" is 6617, and the transcript is NM_003082.4.

[0045] In this article, the "RETSAT gene" has the Gene ID: 54884 and transcript: NM_017750.4.

[0046] In this article, the gene ID of the “P2RY10 gene” is 27334, and the transcript is NM_014499.4.

[0047] In this article, the Gene ID of “Tmem159” is 233806, and the transcript is NM_145586.1. “Teme159” and “TMEM159” are used interchangeably.

[0048] In this Chinese text, "background gene" refers to the gene corresponding to the fluorescence value used in the denominator of the correction formula during fluorescence correction in isothermal fluorescence detection. This gene is usually a gene whose expression is relatively constant in most cells.

[0049] In this article, the Gene ID of “GAPDH” is 2597, and the transcript is NM_002046.7.

[0050] Given the shortcomings of existing technologies, it is essential to develop new, high-performance methods for early screening of preterm births.

[0051] According to a first aspect, in one embodiment, a polynucleotide is provided, the polynucleotide comprising a first anchoring sequence and a first guide sequence connected in tandem, the first anchoring sequence being bindable to the nuclease, the first guide sequence being specific to a target nucleic acid molecule and specifically binding to the target nucleic acid molecule, the first anchoring sequence binding to the nuclease, and the first guide sequence binding to the target nucleic acid molecule causing the target nucleic acid molecule on the target nucleic acid molecule to be cleaved.

[0052] In one embodiment, the first guide sequence is specific to at least one of the genes shown in Table 1 and the background genes.

[0053] In one embodiment, the first guide sequence is specific to at least one of the LMCD1 gene, SNAPC1 gene, RETSAT gene, P2RY10 gene, TMEM159 gene, and background gene.

[0054] In one embodiment, the first guide sequence is specific to at least one of the LMCD1 gene, SNAPC1 gene, RETSAT gene, and GAPDH gene.

[0055] In one embodiment, the first guide sequence contains at least one of the following base sequences:

[0056] 1)5'-ACACAGGAAUCCAUUACAUU-3';

[0057] 2)5'-AGUGGAACAGAGUUCACUGC-3';

[0058] 3)5'-CAAGACUUGCAUAUUUUCCU-3';

[0059] 4)5'-CUGGUAUGACAACGAAUUUG-3'.

[0060] According to a second aspect, in one embodiment, a kit is provided comprising the polynucleotide described in the first aspect.

[0061] In one embodiment, the kit further comprises a nuclease and crRNA, the crRNA comprising the polynucleotide described in the first aspect.

[0062] In one embodiment, the kit further comprises a second guide RNA, a first sequence with a first vacuolar sequence, and a second sequence with a second vacuolar sequence. The second guide RNA comprises a second anchoring sequence, a third binding sequence, and a fourth binding sequence connected in series. The second anchoring sequence comprises the first binding sequence and the second binding sequence. The second anchoring sequence can bind to a nuclease, causing the target nucleic acid molecule on the target nucleic acid molecule to be cleaved.

[0063] In one embodiment, the first sequence includes a first cavitation sequence, a fifth binding sequence concatenated to one end of the first cavitation sequence, and a sixth binding sequence concatenated to the other end of the first cavitation sequence;

[0064] The second sequence includes a second cavitation sequence, a seventh binding sequence concatenated to one end of the second cavitation sequence, and an eighth binding sequence concatenated to the other end of the second cavitation sequence;

[0065] The first binding sequence of the anchoring sequence can be reverse complementary to the sixth binding sequence of the first sequence, and the second binding sequence of the anchoring sequence can be reverse complementary to the fifth binding sequence of the first sequence.

[0066] The third binding sequence of the second guide RNA is inversely complementary to the eighth binding sequence of the second sequence, and the fourth binding sequence of the second guide RNA is inversely complementary to the seventh binding sequence of the second sequence.

[0067] In one embodiment, one end of the first vacuolated sequence and the second vacuolated sequence are independently modified with a first labeling molecule, and the other end of the first vacuolated sequence and the second vacuolated sequence are independently modified with a second labeling molecule. The first vacuolated sequence and the second vacuolated sequence can be cleaved from the first sequence and the second sequence by the nuclease. Before cleavage, the fluorescent reporter group and the fluorescent quencher group are physically close, resulting in fluorescence quenching. After cleavage, the fluorescent reporter group and the fluorescent quencher group are physically far apart, and a strong fluorescence signal can be detected at the excitation wavelength of the fluorescent reporter group.

[0068] In one embodiment, the second guide RNA is modified with a third marker molecule.

[0069] In one embodiment, the second guide RNA is a single-stranded RNA, and the first sequence with a first vacuolar sequence and the second sequence with a second vacuolar sequence are independently single-stranded DNA.

[0070] In one embodiment, the second guide RNA can react with a first sequence carrying a first vacuolated sequence and a second sequence carrying a second vacuolated sequence to obtain a guide RNA (i.e., scgRNA) that can be cleaved by nucleases.

[0071] In one embodiment, the polynucleotide further comprises a product obtained by reacting the second guide RNA with a first sequence carrying a first vacuolated sequence and a second sequence carrying a second vacuolated sequence. This product is a guide RNA (i.e., scgRNA) that can be cleaved by nucleases.

[0072] In one embodiment, the kit further comprises an auxiliary nucleic acid molecule.

[0073] In one embodiment, the helper nucleic acid molecule comprises a base sequence that can be reverse complementary to a portion of the base sequence in the second guide RNA.

[0074] In one embodiment, the helper nucleic acid molecule comprises a base sequence that can be reverse complementary to at least one of the third binding sequence and the fourth binding sequence of the second guide RNA.

[0075] In one embodiment, the helper nucleic acid molecule comprises base sequences that can be reverse complementary to all of the third and fourth binding sequences of the second guide RNA.

[0076] In one embodiment, the auxiliary nucleic acid molecule further comprises a PAM sequence that can be recognized by the nuclease.

[0077] In one embodiment, the auxiliary nucleic acid molecule is double-stranded DNA.

[0078] In one embodiment, the first anchoring sequence and the second anchoring sequence independently contain the following base sequences:

[0079] 5'-UAAUUUCUACUAAGUGUAGAU-3'.

[0080] In one embodiment, the first marker molecule and the third marker molecule are the same type of marker molecule.

[0081] In one embodiment, the first and third labeled molecules are fluorescent reporter groups.

[0082] In one embodiment, the second labeling molecule is a fluorescence quencher group.

[0083] In one embodiment, fluorescence energy resonance transfer occurs when the first labeled molecule and the third labeled molecule are physically close together, and fluorescence energy resonance transfer occurs when the second labeled molecule and the third labeled molecule are physically close together, which may specifically be fluorescence quenching.

[0084] In one embodiment, the fluorescent reporter group includes, but is not limited to, at least one of FAM, HEX, VIC, ROX, and Cy5.

[0085] In one embodiment, the fluorescence quenching group is selected from at least one of BHQ1 and BHQ2.

[0086] In one embodiment, the kit further comprises crRNA specific to at least one of the LMCD1 gene, SNAPC1 gene, RETSAT gene, P2RY10 gene, and TMEM159 gene.

[0087] In one embodiment, the kit further comprises crRNA that is specific to background genes.

[0088] In one embodiment, the background gene includes, but is not limited to, the GAPDH gene.

[0089] In one embodiment, the nuclease includes, but is not limited to, at least one of Cas9 protein and Cas12a protein. Nucleases can be commercially available or obtained through recombinant expression, protein purification, or other methods.

[0090] In one embodiment, the Cas12a protein includes, but is not limited to, at least one of LbCas12a, AsCas12a, and FnCas12a.

[0091] In one embodiment, the crRNA is a single-stranded RNA.

[0092] In one embodiment, the kit also contains other reagents required for the enzyme digestion reaction, including but not limited to buffer, KCl, MgCl2, DTT (dithiothreitol), glycerol, water, etc.

[0093] In one embodiment, the target nucleic acid molecule contains a PAM sequence (protospacer adjacent mot) for recognition by the nuclease.

[0094] In one embodiment, the PAM sequence is typically TTTN, and the cleavage target is typically located downstream of the PAM sequence.

[0095] In one embodiment, the first anchoring sequence is located upstream of the first guide sequence. That is, the 3' end of the first anchoring sequence is connected in series to the 5' end of the first guide sequence.

[0096] According to a third aspect, in one embodiment, the use of at least one of the genes shown in Table 1 as a biomarker in the preparation and / or screening of gestational age prediction reagents is provided.

[0097] In one embodiment, the biomarker includes, but is not limited to, at least one of the following genes: LMCD1, SNAPC1, RETSAT, P2RY10, and TMEM159.

[0098] In one embodiment, the biomarker includes, but is not limited to, at least one of the LMCD1 gene, SNAPC1 gene, and RETSAT gene.

[0099] In one embodiment, the biomarker includes all of the LMCD1 gene, SNAPC1 gene, and RETSAT gene.

[0100] In one embodiment, the biomarker includes the expression profile of the corresponding gene in the maternal sample.

[0101] In one embodiment, the expression profile includes the RNA expression level of the corresponding gene in the maternal sample.

[0102] In one embodiment, the RNA expression level refers to the mRNA expression level. Typically, this is achieved by first obtaining a set of all reverse transcription products through reverse transcription, and then calculating the mRNA expression level.

[0103] In one embodiment, the expression profile includes the protein expression levels of the corresponding gene in the maternal sample.

[0104] In one embodiment, the expression profile includes overexpression of the corresponding gene relative to baseline in the maternal sample to be tested.

[0105] In one embodiment, the expression profile includes underexpression of corresponding genes in the maternal sample to be tested relative to baseline. In some embodiments, a subset of biomarkers in the maternal sample to be tested may be overexpressed relative to baseline, while another subset may be underexpressed relative to baseline. In other embodiments, all biomarkers in the maternal sample to be tested may be overexpressed relative to baseline. In still other embodiments, all biomarkers in the maternal sample to be tested may be underexpressed relative to baseline.

[0106] In one embodiment, the baseline may be the expression profile of the corresponding gene in a maternal sample from a full-term delivery.

[0107] In one embodiment, the expression profile is determined by measuring cell-free RNA (cfRNA) in the maternal sample.

[0108] In one embodiment, the expression profile is determined by measuring proteins in a parent sample.

[0109] In one embodiment, the maternal sample includes, but is not limited to, at least one of bodily fluid samples and tissue samples. Bodily fluid samples are preferred.

[0110] In one embodiment, the bodily fluid sample includes, but is not limited to, at least one of maternal blood, plasma, serum, and urine.

[0111] In one embodiment, the maternal sample is taken between the 7th and 20th weeks of gestation.

[0112] In one embodiment, the biomarker includes all of the LMCD1 gene, SNAPC1 gene, and RETSAT gene.

[0113] In one embodiment, the biomarker is the fluorescence value of the product after the corresponding gene is digested by an enzyme in a system containing a fluorescent reagent.

[0114] In one embodiment, the enzymatic digestion reaction is an isothermal reaction. That is, the temperature of the reaction system is kept constant during the enzymatic digestion reaction.

[0115] In one embodiment, the temperature of the enzymatic digestion reaction is 35–42°C, preferably 37°C.

[0116] In one embodiment, fluorescence values ​​are collected at a certain time point or during a certain period of time during the enzyme digestion reaction;

[0117] In one embodiment, the fluorescence value is collected at a certain time point or time period between 30 and 120 minutes of the enzyme digestion reaction.

[0118] In one embodiment, the fluorescence value is collected at the 90th minute of the enzyme digestion reaction. This time includes the time required for the temperature to rise from room temperature to the target temperature.

[0119] In one embodiment, the starting point of the enzymatic digestion reaction is typically when the reactor is started. The heating rate from room temperature (23±2℃) to 37℃ is typically 4℃ / s.

[0120] In one embodiment, the biomarker is the fluorescence value of the reverse transcription product of the corresponding gene RNA in the maternal sample after enzymatic digestion in a system containing a fluorescent reagent.

[0121] In one embodiment, the enzyme digestion reaction system further contains the polynucleotide described in the first aspect or the components of the kit described in the second aspect.

[0122] In one embodiment, the probability of premature birth in a subject is calculated by analyzing the expression profile of the corresponding gene in the maternal sample.

[0123] In one embodiment, the probability of premature birth in the subject is calculated using the expression profile of corresponding genes in the maternal sample. Based on the relationship between the probability value and a threshold, the likelihood of premature birth is predicted. In one embodiment, the threshold can be selected as the threshold that maximizes the sum of sensitivity and specificity in the training set.

[0124] In one embodiment, the threshold is 0.4 to 0.7. In another embodiment, the threshold is 0.45.

[0125] In one embodiment, if the probability value is greater than or equal to a threshold, the subject is predicted to be premature; if the probability value is less than the threshold, the subject is predicted to be full-term.

[0126] In one embodiment, the probability of premature birth in a subject is calculated using the fluorescence value of the corresponding gene.

[0127] In one embodiment, the probability of premature birth in the subject is calculated using the fluorescence correction value of the corresponding gene. The purpose of the correction is to avoid the calculated value being too small.

[0128] In one embodiment, the fluorescence value is the fluorescence correction value.

[0129] In one embodiment, the fluorescence correction value of the reverse transcription product of the corresponding gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent is calculated as follows: the fluorescence value of the reverse transcription product of the corresponding gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent is divided by the fluorescence value of the reverse transcription product of the background gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent to obtain a quotient value, and the quotient value is multiplied by a correction coefficient to obtain the fluorescence correction value.

[0130] In one embodiment, the correction factor includes, but is not limited to, 10^5. The correction factor can be any other arbitrary constant, which can be determined as needed, with the aim of avoiding the calculated value being too small.

[0131] In one embodiment, the formula for calculating the probability of premature birth in a subject is as follows: The formula is based on the fluorescence correction value of the product obtained after enzyme digestion of the reverse transcription product of the corresponding gene RNA in the maternal sample in a system containing fluorescent reagent.

[0132] Wherein, the F LMCD1 The F value is the fluorescence correction value of the reverse transcription product of the LMCD1 gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent. SNAPC1 The F value is the fluorescence correction value of the reverse transcription product of the SNAPC1 gene RNA from the maternal sample after enzymatic digestion in a system containing fluorescent reagent. RETSAT The fluorescence correction value is the product of the reverse transcription product of the RET SAT gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent.

[0133] According to the fourth aspect, in one embodiment, a method for predicting preterm birth is provided, comprising: analyzing the expression profile of any one or at least two genes in a maternal sample, and predicting whether the subject will be premature based on the expression profile.

[0134] It should be noted that the method for predicting preterm birth provided by this invention detects in vitro samples, and the prediction results are only for clinical reference. When performing preterm birth detection and / or diagnosis, doctors also need to use clinical estimation (usually based on previous menstrual cycles), ultrasound examination (e.g., fetal head diameter, head circumference, abdominal circumference, and femur length are closely related to gestational age and weight; gestational age and fetal size can be estimated based on ultrasound measurements), and fetal fibronectin (fFN) swab testing (fetal fibronectin is jointly secreted by the amnion, decidua, and chorionic villi, and is stored in...). The glycoproteins located between the decidua and chorion play a role in the adhesion of the fetal membranes. After 21 weeks of gestation, the fusion of the chorion and decidua prevents the release of fetal fibrinogen (fFN). Therefore, in normal pregnant women, the fFN content is extremely low between 22 and 35 weeks of gestation. FFN leaks into the posterior fornix secretions of the vagina when the chorion and decidua separate, when the extracellular matrix at the chorion-decidua interface is mechanically damaged, or when it is degraded by proteolytic enzymes. (The fFN level in cervical and vaginal secretions between 22 and 35 weeks of gestation is strongly correlated with preterm labor.) Only through other means, such as these, can a final result be determined regarding whether preterm labor has occurred. Therefore, the predictive results of this invention are not final results, but rather intermediate reference results, and are not a diagnostic method for the disease, much less a treatment method.

[0135] In one embodiment, the gene includes at least one of the genes shown in Table 1.

[0136] In one embodiment, the gene includes, but is not limited to, at least one of the following: LMCD1 gene, SNAPC1 gene, RETSAT gene, P2RY10 gene, and TM EM159 gene;

[0137] In one embodiment, the gene includes at least one of the LMCD1 gene, the SNAPC1 gene, and the RETSAT gene.

[0138] In one embodiment, the genes include all of the LMCD1 gene, SNAPC1 gene, and RETSAT gene.

[0139] In one embodiment, the expression profile is the expression level of the corresponding gene in the maternal sample.

[0140] In one embodiment, the expression profile is the fluorescence value of the corresponding gene after enzyme digestion.

[0141] In one embodiment, the expression profile is the fluorescence value of the reverse transcription product of the corresponding gene's RNA in the maternal sample after enzymatic digestion. This method eliminates the need for sequencing analysis of the maternal sample library, avoids the use of expensive sequencing equipment and reagents, and effectively simplifies the detection process. It eliminates the need for PCR reaction heating and cooling processes, and the need for complex PCR instruments, requiring only a fluorescence reading device.

[0142] In one embodiment, the fluorescence value is the fluorescence correction value.

[0143] In one embodiment, the enzymatic digestion reaction is an isothermal reaction. An isothermal reaction means that the temperature remains constant during the enzymatic digestion reaction, or the temperature fluctuation is minimal.

[0144] In one embodiment, the enzyme digestion reaction system also contains a fluorescent reagent.

[0145] In one embodiment, the enzyme digestion reaction system contains the polynucleotide described in the first aspect or the components of the kit described in the second aspect.

[0146] According to a fifth aspect, in one embodiment, an isothermal enzyme digestion detection method is provided, comprising: performing an enzyme digestion reaction on a template to be tested under isothermal conditions, and collecting fluorescence values ​​during and / or at the end of the enzyme digestion reaction. This method can be used for the detection of the relative expression level of a target gene, or for the qualitative detection of a target sequence. This enzyme digestion detection method has advantages such as quantitative detection, isothermal detection, high sensitivity, and ease of operation.

[0147] In one embodiment, the fluorescence value is collected at a certain time point or time period between the 30th and 120th minute.

[0148] In one embodiment, the fluorescence value is collected at the 90th minute.

[0149] In one embodiment, the temperature of the enzymatic digestion reaction is 35-42°C, including but not limited to 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, etc., preferably 37°C.

[0150] In one embodiment, the enzyme digestion reaction system contains the polynucleotide described in the first aspect or the components of the kit described in the second aspect.

[0151] In one embodiment, the template to be tested can be the reverse transcription product of the RNA of the gene to be tested in the maternal sample.

[0152] In one embodiment, the RNA comprises messenger RNA (mRNA).

[0153] According to a sixth aspect, in one embodiment, a system for predicting preterm birth is provided, comprising: a preterm birth analysis device for analyzing the expression profile of any one or at least two genes in a maternal sample, and predicting whether a subject will be born prematurely based on the expression profile.

[0154] According to a seventh aspect, in one embodiment, an apparatus is provided, comprising:

[0155] Memory, used to store programs;

[0156] A processor for implementing the method described in the fifth aspect by executing a program stored in the memory.

[0157] According to the eighth aspect, in one embodiment, a computer-readable storage medium is provided, the medium storing a program that can be executed by a processor to implement the method as described in the fifth aspect.

[0158] In one embodiment, the kit provided by the present invention is suitable for enzymatic digestion and fluorescence detection of the reverse transcription product of the RNA of the gene to be tested in the maternal sample as the template for the test.

[0159] In one embodiment, the kit is a kit for predicting preterm birth in pregnant women.

[0160] In one embodiment, the present invention provides a quantitative detection method for early screening of preterm birth in humans based on the expression levels of LMCD1, SNAPC1 and RETSAT genes.

[0161] In one embodiment, this invention first detects the expression levels of LMCD1, SNAPC1, and RETSAT genes in the serum of pregnant women for the prediction of preterm birth risk in asymptomatic pregnant women. The main advantages are that the serological testing does not require prior appointment like ultrasound, it is non-invasive, does not require vaginal or cervical sampling, and offers a better user experience. The detection uses immunochromatography, which is simple and low-cost. It has high sensitivity and specificity, allows for repeated monitoring, and has better clinical application prospects compared to traditional methods.

[0162] In one embodiment, the present invention predicts the risk of preterm birth based on the expression levels of detected mRNA biomarkers using machine learning. Positive samples are provided to clinicians as supplementary diagnostic aids.

[0163] In one embodiment, the detection principle of the present invention is as follows: the expression levels of LMCD1, SNAPC1 and RETSAT genes in the serum of pregnant women can reflect the risk of premature birth in pregnant women. This detection is not affected by whether the pregnant woman has relevant clinical symptoms and has no contraindications. The risk of asymptomatic premature birth can be predicted by monitoring the expression levels of LMCD1, SNAPC1 and RETSAT genes.

[0164] In one embodiment, the detection reagent provided by the present invention is targeted at the peripheral blood of pregnant women.

[0165] In one embodiment, the sample collection for the detection of the present invention has no contraindications and can be performed multiple times.

[0166] In one embodiment, the mRNAs detected by the present invention are LMCD1, SNAPC1, and RETSAT.

[0167] In one embodiment, the fluorescence detection based on the enzyme digestion reaction of the present invention can be completed under fixed conditions. During the enzyme digestion reaction, after heating to the target temperature, there is no need for a heating and cooling process.

[0168] In one embodiment, the present invention provides a plugin for comparative analysis of the levels of three biomarkers, automatically generating risk values. The plugin includes a formula for calculating the probability of premature birth in a subject based on the fluorescence correction value of the product obtained after enzymatic digestion of the reverse transcription product of the corresponding gene's RNA in a system containing fluorescent reagents, as detailed below:

[0169] Wherein, the F LMCD1 The F value is the fluorescence correction value of the reverse transcription product of the LMCD1 gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent. SNAPC1 The F value is the fluorescence correction value of the reverse transcription product of the SNAPC1 gene RNA from the maternal sample after enzymatic digestion in a system containing fluorescent reagent. RETSAT The fluorescence correction value is the product of the reverse transcription of the RETSAT gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent.

[0170] In one embodiment, the high sensitivity and high specificity of the present invention can effectively improve the accuracy of preterm birth screening in pregnant women and make up for the shortcomings of existing clinical methods.

[0171] In one embodiment, the present invention uses peripheral blood from pregnant women for testing, which is non-invasive and does not require vaginal or cervical sampling, making it convenient for clinical sampling and testing.

[0172] In one embodiment, the present invention employs an isothermal high-sensitivity detection method based on CAS12A, which has low requirements for detection conditions, is simple, and has low cost.

[0173] In one embodiment, the present invention can provide a method for assessing the risk of premature birth for asymptomatic pregnant women, and can screen out pregnant women at high risk of premature birth and take measures such as intrauterine transfer in advance, thereby reducing the risk of premature birth or improving the survival rate of premature infants, which is of great significance for increasing the birth population in my country.

[0174] In the following examples, the first day of the last menstrual period before pregnancy is taken as the start of pregnancy.

[0175] Example 1: Sampling and Plasma Separation

[0176] Collect 5 mL of blood using an EDTA anticoagulant tube (purple-headed tube). After collection, temporarily store the blood at 4°C and then have it centrifuged by the relevant personnel or laboratory within 8 hours to collect plasma and white blood cells. Then, quickly freeze the blood at -80°C.

[0177] The specific steps for plasma separation are as follows:

[0178] 1) After inverting each blood collection tube 10 times, place it in a pre-cooled large centrifuge.

[0179] 2) Centrifuge at 4℃, 1600×g for 10 minutes.

[0180] 3) Carefully remove the blood collection tube from the centrifuge and place it back on the 15mL centrifuge tube rack.

[0181] 4) After centrifugation, the blood sample is divided into a bottom red blood cell layer, a middle white blood cell layer, and a supernatant plasma layer (normal plasma is pale yellow, and there may be some white flocculent matter at the top of this layer, which is lipid substance).

[0182] 5) Adjust the 1000μL pipette to 500μL, carefully transfer the plasma into 1.5mL centrifuge tubes, 500μL per tube, and finally transfer the leukocytes in the middle layer into 1.5mL centrifuge tubes.

[0183] 6) Place 3 tubes of plasma into a small centrifuge and centrifuge at 16000×g for 10 minutes at room temperature (the main purpose is to remove cell debris, etc.).

[0184] 7) Adjust the 1000μL pipette to 500μL and carefully transfer the plasma into a 1.5mL centrifuge tube.

[0185] 8) Both plasma and leukocytes were stored at -80°C for a long period of time. Leukocytes were not used in subsequent examples.

[0186] Example 2: cfRNA extraction

[0187] 1) Centrifuge plasma samples at a centrifugal force of ≥12,000×g for 15 minutes to remove cellular impurities and precipitates.

[0188] 2) Add 200 μL of free RNA digestion solution to every 200 μL of sample, place in a 15 mL centrifuge tube, and mix well. If the sample volume is ≥1.5 mL, use a 50 mL centrifuge tube. The free RNA digestion solution, proteinase K solution, RNA solution 1, and RNA solution 2 are contained in one kit, purchased from Beijing Tianmo Technology Development Co., Ltd., catalog number TR159-50.

[0189] 3) Add 10 μL of proteinase K solution to every 200 μL of plasma sample, vortex for 10 seconds, and incubate at 37°C for 2 hours.

[0190] 4) Add an equal volume of free RNA binding solution to the digested sample and vortex mix for 10 seconds.

[0191] 5) Add 1.5 times the volume of 100% isopropanol to the mixture in step 4) and vortex mix for 10 seconds.

[0192] 6) Fit the 25mL funnel into column Y (yellow) No. 3, connect it tightly, and place it on the negative pressure multi-connector.

[0193] 7) Pour in the mixture from step 6), turn on the vacuum switch to allow the liquid to pass completely through the column. After ensuring that the liquid has completely passed through the column and there is no residual liquid flowing down, turn off the vacuum pump and disconnect the connected 25mL funnel.

[0194] 8) Pour 600 μL of RNA washing buffer 1 onto the column, turn on the vacuum switch, and allow the liquid to completely pass through column Y (yellow) of column 3. Then turn off the vacuum pump.

[0195] 9) Place column Y (yellow) on a 2mL collection tube, then centrifuge at full speed for 2 minutes on a benchtop centrifuge to remove any liquid residue. Then place column Y (yellow) on a clean 1.5mL centrifuge tube.

[0196] 10) Add 200 μL of RNA recovery buffer to column Y (yellow) of column 3, incubate at room temperature for 3 minutes, centrifuge, and save the filtrate.

[0197] 11) Add 300 μL of anhydrous ethanol (95-100%) to the filtrate and mix well.

[0198] 12) Attach column 1 to a new 2 mL collection tube. Add the mixture obtained in step 11) to the column and centrifuge to remove the filtrate.

[0199] 13) Add 400 μL of RNA washing buffer 1, centrifuge to remove the filtrate.

[0200] 14) Add 700 μL of RNA washing buffer 2, centrifuge to remove the filtrate.

[0201] 15) Add 400 μL of RNA washing buffer to column 1, centrifuge for 2 minutes to completely remove any residual liquid from the column, and transfer column 1 to a clean centrifuge tube.

[0202] 16) Add 15 μL of RNase-free water to the column matrix, incubate for 2 minutes and then centrifuge to elute free RNA.

[0203] Example 3: Screening for biomarkers using transcriptome sequencing

[0204] 1. Thirty-nine preterm birth cases were collected during early pregnancy (8-20 weeks), and peripheral blood samples were collected from 42 full-term cases. Cell-free RNA was extracted from peripheral serum of all samples and subjected to whole transcriptome sequencing. This involves sequencing all intracellular transcripts, including messenger RNA, ribosomal RNA, transfer RNA, and non-coding RNA, etc., and then further screening for target messenger RNA (mRNA).

[0205] Figure 1 The image shows the distribution of premature delivery times. Figure 2 The image shows the distribution of full-term delivery times. Figure 3 The image shows the distribution of sampling times for premature births. Figure 4 The diagram shows the timeline for full-term sample collection.

[0206] 2. Differentially expressed genes were screened using DEseq2, with a p-value less than 0.05 as the threshold. A total of 935 differentially expressed genes were identified, as shown in Table 1. Differential expression here refers to the presence of differentially expressed genes in samples from preterm deliveries compared to full-term deliveries, including both high and low expression relative to baseline.

[0207] Table 1

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214] 3. For the TPM data, the R software caret package was used to screen for the most important RNA biomarkers using a random forest algorithm with 10-fold cross-validation. Ultimately, five RNA biomarkers (mRNA) corresponding to the genes were selected. The five genes are P2RY10, LMCD1, SNAPC1, RET SAT, and Tmem159.

[0215] 4. Logistic regression was used to perform regression analysis on the five genes, and the three most important RNA markers (including only mRNA) were finally identified, namely LMCD1, SNAPC1 and RETSAT mRNA, which can be used to predict preterm birth.

[0216] Figure 5The figure shows the expression levels of LMCD1, SNAPC1 and RETSAT genes in each group. It can be seen that LMCD1 and SNAPC1 genes are underexpressed relative to full-term samples, while RETSAT gene is overexpressed relative to full-term samples.

[0217] Figure 6 The figure shows the ROC curve (receiver operating characteristic curve) of the training set, where the AUC (Area Under Curve) is 0.908.

[0218] Figure 7 The figure shows the ROC curve of the test set, where the AUC (Area Under Curve) is 0.846.

[0219] Example 4: Reverse Transcription

[0220] The reverse transcription system is as follows:

[0221] Table 2

[0222]

[0223] Reverse transcription can be performed using either of the two systems in Table 2. In this example, the 20 μL system was specifically selected.

[0224] The kit used in Table 2 is AKTaqOne-StepRT-PCRMix purchased from FAPON, product number: MD103.

[0225] The processing steps are shown in the table below:

[0226] Table 3

[0227] step temperature time 1 42℃ 5min 2 95℃ 10s

[0228] Example 5: Isothermal fluorescence detection

[0229] In this embodiment, the nucleotide molecules used for isothermal fluorescence detection were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0230] For the genes LMCD1, SNAPC1, and RETSAT that need to be detected, the corresponding Cas12a crRNA (C RISPR-derived RNA) sequences were designed and optimized using AHCB as follows:

[0231] Table 4

[0232]

[0233] In Table 4, the gRNA specifically targeting the RETSAT gene guides LbCas12a enzyme cleavage, specifically by cutting the cDNA sequence generated from the reverse-transcribed RETSAT mRNA, initiating a positive feedback cascade reaction. The other three gRNAs function similarly to the RETSAT gene gRNA. The cDNA resulting from the reverse transcription of the mRNA of the genes listed in Table 4 contains a PAM sequence for recognition by the LbCas12a enzyme; the PAM sequence is specifically TTTN.

[0234] In Table 4, the bolded bases are the first anchoring sequences. The unbolded bases are the first guide sequences that can bind to the target gene.

[0235] The principle of enzyme digestion reaction is as follows: Figure 14 As shown, the gRNA targeting the target sequence guides cas12a to cleave the target sequence, generating non-specific cleavage activity. The cleavage vacuoles produce fluorescence, and at the same time, the RNA bound to the two iDNAs releases gRNA targeting the helper double-stranded DNA, which guides cas12a to cleave the helper double-stranded DNA and activates non-specific cleavage activity, further cleaving the RNA (scgRNA) bound to the two iDNAs, generating fluorescence. This produces a positive feedback amplification signal.

[0236] gRNA-FAM sequence: FAM is a green fluorescent reporter group, its English name is 6-carboxyfluorescein, and its Chinese name is 6-carboxyfluorescein. The gRNA-FAM sequence is used to generate a self-feedback amplification effect. The bolded bases are the second anchoring sequences, the dotted-short underlined sequences are the first binding sequences of the second anchoring sequences, the double underlined sequences are the second binding sequences of the second anchoring sequences, the single underlined sequence is the third binding sequence, and the single dashed underlined sequence is the fourth binding sequence.

[0237] The vacuolated sequence 1 (iDNA1) that binds to gRNA-FAM: The sequence marked with a dot-short underline (i.e., the sixth binding sequence) can pair in reverse complement to the sequence marked with a dot-short underline in the gRNA-FAM sequence. The sequence marked with a double straight underline (i.e., the fifth binding sequence) can pair in reverse complement to the sequence marked with a double straight underline in the gRNA-FAM sequence. The sequence marked with a single wavy line in the middle is the first vacuolar sequence, which cannot pair in complement to the gRNA-FAM sequence and is used to generate a fluorescent signal after being cleaved.

[0238] Sequence 2 (iDNA2) with vacuoles that binds to gRNA-FAM: The sequence indicated by a single underline (i.e., the eighth binding sequence) can be reverse complementary to the sequence indicated by a single underline in the gRNA-FAM sequence, and the sequence indicated by a single dashed underline (i.e., the seventh binding sequence) can be reverse complementary to the sequence indicated by a single dashed underline in the gRNA-FAM sequence.

[0239] In iDNA1 and iDNA2, "i6-FAMdT" refers to dT nucleotides labeled with 6-FAM (6-carboxyfluorescein) fluorescence (d indicates deoxy), and "iBHQ1dT" refers to dT nucleotides labeled with the BHQ1 quencher group. dT nucleotides are deoxynucleosides, and "d" is an abbreviation for "deoxidized," where "de" is a prefix indicating removal or detachment, and "oxidize" refers to oxidation. BHQ1 refers to a black hole quenching group. iDNA1 and iDNA2 are used to generate fluorescence after cleavage.

[0240] Auxiliary double-stranded DNA (aDNA) sequence: The sequence indicated by a single dashed underline (the ninth binding sequence) can be reverse complementary to the sequence indicated by a single dashed underline in the gRNA-FAM sequence, and the sequence indicated by a single straight underline (i.e., the tenth binding sequence) can be reverse complementary to the sequence indicated by a single straight underline in the gRNA-FAM sequence.

[0241] iDNA1, iDNA2, and gRNA-FAM generate scgRNA (self-cutting guide RNA) through the following steps:

[0242] iDNA1, iDNA2, and gRNA-FAM were mixed at a molar ratio of 1.2:1 (i.e., the molar ratio of iDNA1 to gRNA-FAM was 1.2:1, and the molar ratio of iDNA2 to gRNA-FAM was also 1.2:1). The mixture was then treated at 95°C for 4 minutes in an annealing buffer (20 mM tris-HCl (pH 7.5), 150 mM KCl, 1 mM EDTA, and 50 mM MgCl2), and then cooled to 25°C at a rate of 0.1°C per second to obtain scgRNA. This product was used as the reactant in Table 4 for subsequent reactions.

[0243] Then prepare the system shown in Table 5.

[0244] Table 5

[0245] Components Final concentration Reverse transcription template DNA 500nM gRNA-T 50nM LbCas12a (purchased from NEB) 1μM scgRNA 1μM aDNA 0.5μM tris-HCl (pH 7.5) 20mM KCl 100mM <![CDATA[MgCl2]]> 5mM DTT (dithiothreitol) 1mM glycerin 5% (by volume) Ultrapure water Make up to 20 μL Total volume 20μL

[0246] In Table 5, gRNA-T refers to one of the crRNAs shown in Table 4 ("T" is short for Target, and gRNA-T means gRNA that targets the target gene, i.e. crRNA). A total of 4 systems were prepared, each system was put into a reaction tube, and each system contained one gRNA, resulting in systems containing four gRNAs.

[0247] The above system was added to PCR tubes, divided into 4 tubes, each containing one of the crRNAs listed in Table 4. The reaction was carried out in a Thermo Fisher Scientific ABI 7500 real-time quantitative PCR instrument (ABI 7500 for short), with the temperature set at 37℃. The temperature was increased from room temperature (23±2℃) to 37℃ at a rate of 4℃ / s. Fluorescence values ​​were collected every 30 seconds, the emission wavelength was 520nm, and the reaction time was 90 minutes.

[0248] Example 6: Data Analysis

[0249] After reacting for 90 minutes in an ABI 7500, fluorescence values ​​were collected as the final expression levels. The fluorescence values ​​of RETSAT, SNAPC1, and LMCD1 (since the reactions were performed in different PCR tubes, the fluorescence values ​​of each RNA marker could be obtained individually) were divided by the fluorescence value of GAPDH, and then multiplied by 10^5 to obtain the final relative expression levels. As the enzyme digestion reaction proceeds, the increase in fluorescence values ​​typically goes through a baseline phase, an exponential phase, a linear phase, and a plateau phase. In this example, the fluorescence value was collected at the 90-minute mark during the exponential growth phase.

[0250] Example 7: Clinical Sample Validation Performance

[0251] 125 full-term samples and 123 preterm samples were collected. The gestational age at delivery and the gestational age at sample collection are as follows: Figures 8-11 shown, specifically, Figure 8 The chart shown is a statistical chart of gestational weeks at full-term delivery. Figure 9 The chart shown is a statistical chart of gestational weeks for premature births. Figure 10 The chart shown is a statistical chart of gestational weeks at full term. Figure 11 The chart shown is a statistical chart of gestational weeks for samples from preterm births.

[0252] cfRNA was extracted and fluorescence measured. The fluorescence values ​​are shown in the table below:

[0253] Table 6

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261] The fluorescence values ​​of each sample gene in Table 5 are the original values ​​collected (without correction).

[0262] The samples were randomly divided into a test set and a training set in a 7:3 ratio. The training set was used to train the model, and the test set was used to validate the model. Figure 12 The image shows the ROC curve for the training set, with an AUC of 0.838. Figure 13 The image shows the ROC curve for the test set, with an AUC of 0.849. At a threshold of 0.450, the training set sensitivity is 0.753 and specificity is 0.765, while the test set sensitivity is 0.711 and specificity is 0.889. The probability calculation formula is as follows:

[0263] Wherein, FLMCD1 is the fluorescence correction value of the reverse transcription product of the LMCD1 gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent; FSNAPC1 is the fluorescence correction value of the reverse transcription product of the SNAPC1 gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent; and FRETSAT is the fluorescence correction value of the reverse transcription product of the RETSAT gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent.

[0264] Those skilled in the art will understand that all or part of the functions of the various methods in the above embodiments can be implemented by hardware or by computer programs. When all or part of the functions in the above embodiments are implemented by computer programs, the program can be stored in a computer-readable storage medium, which may include: read-only memory, random access memory, disk, optical disk, hard disk, etc., and the program is executed by a computer to achieve the above functions. For example, the program can be stored in the memory of a device, and when the program in the memory is executed by the processor, all or part of the above functions can be achieved. In addition, when all or part of the functions in the above embodiments are implemented by computer programs, the program can also be stored in a server, another computer, disk, optical disk, flash drive, or external hard drive, etc., and can be downloaded or copied to the memory of a local device, or the system of the local device can be updated. When the program in the memory is executed by the processor, all or part of the functions in the above embodiments can be achieved.

[0265] The above examples illustrate the present invention only to aid in understanding it and are not intended to limit the scope of the invention. Those skilled in the art can make various simple deductions, modifications, or substitutions based on the principles of this invention.

Claims

1. The use of a reagent for detecting the expression level of biomarker mRNA in maternal samples in the preparation of reagents for predicting preterm birth, characterized in that, The maternal sample includes peripheral blood cfRNA. The biomarker consists of the LMCD1 gene, SNAPC1 gene, and RETSAT gene. A probability value for preterm birth is calculated. If the probability value is greater than or equal to a threshold, preterm birth is predicted; if the probability value is less than the threshold, full-term delivery is predicted. The threshold is 0.

45. The probability value is calculated as follows: Calculate the fluorescence correction value of the reverse transcription product of the corresponding gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent: divide the fluorescence value of the reverse transcription product of the corresponding gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent by the fluorescence value of the reverse transcription product of the background gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent to obtain a quotient value, and multiply the quotient value by a correction coefficient to obtain the fluorescence correction value; The correction coefficient is 10^5; The probability of premature birth in a subject is calculated by measuring the fluorescence correction value of the reverse transcription product of the corresponding gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent, using the following formula: , wherein, the F LMCD1 The F value is the fluorescence correction value of the reverse transcription product of the LMCD1 gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent. SNAPC1 The F value is the fluorescence correction value of the reverse transcription product of the SNAPC1 gene RNA from the maternal sample after enzymatic digestion in a system containing fluorescent reagent. RETSAT The fluorescence correction value is the product of the reverse transcription of the RETSAT gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent.

2. The use as described in claim 1, characterized in that, The maternal samples were collected between the 8th and 20th weeks of gestation.

3. The use as described in claim 1, characterized in that, The enzymatic digestion reaction is an isothermal reaction; And / or, the temperature of the enzymatic digestion reaction is 35~42℃.

4. The use as described in claim 3, characterized in that, The enzyme digestion reaction was performed at a temperature of 37°C.

5. The use as described in claim 3 or 4, characterized in that, The fluorescence value was collected at the 90th minute of the enzyme digestion reaction, or at a certain time point or time period between the 90th and 120th minute of the enzyme digestion reaction.

6. The use as described in claim 1, characterized in that, The system contains a polynucleotide, which includes a first anchoring sequence and a first guide sequence connected in series. The first anchoring sequence can bind to a nuclease, and the first guide sequence is specific to the target nucleic acid molecule and can specifically bind to the target nucleic acid molecule. The first anchoring sequence binds to the nuclease, and after the first guide sequence binds to the target nucleic acid molecule, the target nucleic acid molecule on the target nucleic acid molecule is cleaved.

7. The use as described in claim 6, characterized in that, The first guide sequence is specific to the LMCD1 gene, or the SNAPC1 gene, or the RETSAT gene, or the GAPDH gene; And / or, the first guide sequence contains the following base sequence: 1) 5'-ACACAGGAAUCCAUUACAUU-3'; or 2) 5'-AGUGGAACAGAGUUCACUGC-3'; or 3) 5'-CAAGACUUGCAUAUUUUCCU-3'; or 4) 5'-CUGGUAUGACAACGAAUUUG-3'.

8. The use as described in claim 6 or 7, characterized in that, The system also contains nucleases; And / or, the system further contains a nuclease, the nuclease including at least one of Cas9 protein and Cas12a protein; And / or, the system further contains Cas12a protein, which includes at least one of LbCas12a, AsCas12a, and FnCas12a.

9. A system for predicting preterm birth, characterized in that, include: A preterm birth analysis device is used to analyze the expression profile of a gene set in a maternal sample and predict whether a subject will be born prematurely based on the expression profile. The maternal sample includes peripheral blood cfRNA, and the gene set consists of the LMCD1 gene, SNAPC1 gene, and RETSAT gene. The device predicts whether a subject will be born prematurely by calculating a probability value of premature birth. If the probability value is greater than or equal to a threshold, the subject is predicted to be born prematurely; if the probability value is less than the threshold, the subject is predicted to be born at full term. The threshold is 0.

45. The probability value is calculated as follows: Calculate the fluorescence correction value of the reverse transcription product of the corresponding gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent: divide the fluorescence value of the reverse transcription product of the corresponding gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent by the fluorescence value of the reverse transcription product of the background gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent to obtain a quotient value, and multiply the quotient value by a correction coefficient to obtain the fluorescence correction value; The correction coefficient is 10^5; The probability of premature birth in a subject is calculated by measuring the fluorescence correction value of the reverse transcription product of the corresponding gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent, using the following formula: , wherein, the F LMCD1 The F value is the fluorescence correction value of the reverse transcription product of the LMCD1 gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent. SNAPC1 The F value is the fluorescence correction value of the reverse transcription product of the SNAPC1 gene RNA from the maternal sample after enzymatic digestion in a system containing fluorescent reagent. RETSAT The fluorescence correction value is the product of the reverse transcription of the RETSAT gene RNA in the maternal sample after enzymatic digestion in a system containing fluorescent reagent.

Images