A system for early screening of neurodevelopmental delay in mid-to-late-term preterm infants

Abstract

This invention discloses a system for early screening of neurodevelopmental delay in mid-to-late-term preterm infants. Based on the expression levels of two mRNAs, APOE and CST3, in the placenta, this invention establishes a "2-mRNA Panel" classification model for early screening of neurodevelopmental delay in mid-to-late-term preterm infants. The area under the receiver operating characteristic curve (AUC) for gross motor delay, fine motor delay, and personal-social functional delay reaches 86.1%, 82.2%, and 83.3%, respectively. Therefore, APOE and CST3 mRNAs in the placenta can serve as biomarkers for neurodevelopmental delay in mid-to-late-term preterm infants, providing a valuable supplement to the development of early screening tools for neurodevelopmental delay. This invention has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a system for early screening of neurodevelopmental delay in mid-to-late-term premature infants. Background Technology

[0002] Mid-to-late premature infants are live births born between 32 and 37 weeks of gestation. Insufficient intrauterine development in mid-to-late premature infants can lead to developmental delays and cause immense emotional distress and financial burden on families and society.

[0003] Early assessment and intervention for neurodevelopmental delay in mid-to-late-term preterm infants can help improve their quality of life. Currently, clinicians mainly use scales to screen for neurodevelopmental delay in children. While these scales have high reliability and validity, most are administered at a relatively late age and only serve an assessment purpose, failing to aid in etiological diagnosis, thus having certain limitations. Therefore, a deeper understanding of the biological mechanisms and biomarkers of neurodevelopmental delay in mid-to-late-term preterm infants is of significant value.

[0004] According to the developmental and origin theory of diseases, adverse experiences during fetal development in utero significantly increase the risk of illness in early life. This suggests that the placenta's response to various intrauterine stressors may influence a child's growth trajectory, and studying placental changes can help understand the mechanisms of neurodevelopmental delay in mid-to-late-term preterm infants. Recent studies have shown that many genes in the placenta affect fetal brain development; these genes are defined as "placental-brain axis genes." With the continuous development of omics technologies in recent years, "placental-brain axis genes" have shown potential in predicting neurodevelopmental delay in mid-to-late-term preterm infants. Summary of the Invention

[0005] The purpose of this invention is to provide an early screening method for neurodevelopmental delay in mid-to-late-term premature infants.

[0006] This invention first protects a system for early screening of neurodevelopmental delay in mid-to-late-term preterm infants, which can detect the expression levels of APOE and CST3 in placental RNA; based on the expression levels of APOE and CST3 in placental RNA, -ΔCt values ​​are obtained, a machine learning model is trained, and a random forest machine learning model is obtained as a classification model, thereby realizing early screening of neurodevelopmental delay in mid-to-late-term preterm infants.

[0007] The system described above, which detects the expression levels of APOE and CST3 in placental RNA, may include reagents required for detecting the relative expression levels of APOE and CST3 in placental RNA using real-time quantitative PCR.

[0008] The system described above for detecting the expression levels of APOE and CST3 in placental RNA can specifically consist of reagents required for detecting the relative expression levels of APOE and CST3 in placental RNA using real-time quantitative PCR.

[0009] In the above system, the reagents required for detecting the relative expression levels of APOE and CST3 in placental RNA using real-time quantitative PCR can be the same reagents required for detecting the relative expression levels of APOE and CST3 in placental RNA relative to the internal control using real-time quantitative PCR. The internal control can be 18S rRNA.

[0010] In the above system, the reagents required for detecting the expression levels of APOE and CST3 relative to the internal reference in placental RNA using the real-time quantitative PCR method may include primer pairs for detecting APOE, primer pairs for detecting CST3, and primer pairs for detecting 18S rRNA.

[0011] In the above system, the reagents required for detecting the expression levels of APOE and CST3 relative to the internal reference in placental RNA using the real-time quantitative PCR method can specifically consist of primer pairs for detecting APOE, primer pairs for detecting CST3, and primer pairs for detecting 18S rRNA.

[0012] The primer pair for detecting APOE can consist of a primer with a nucleotide sequence as shown in SEQ ID NO:1 and a primer with a nucleotide sequence as shown in SEQ ID NO:2.

[0013] The primer pair for detecting CST3 can consist of a primer with a nucleotide sequence as shown in SEQ ID NO:3 and a primer with a nucleotide sequence as shown in SEQ ID NO:4.

[0014] Primer pairs for detecting 18S rRNA can consist of primers with nucleotide sequences as shown in SEQ ID NO:5 and primers with nucleotide sequences as shown in SEQ ID NO:6.

[0015] In any of the systems described above, the neurodevelopmental delay may be gross motor delay, fine motor delay, or personal-social delay.

[0016] In any of the systems described above, the neurodevelopmental delay can be neurodevelopmental delay at 6 months of age.

[0017] This invention also protects the use of reagents required for detecting the relative expression levels of APOE and CST3 in placental RNA using real-time quantitative PCR in the preparation of products for early screening of neurodevelopmental delay in mid-to-late-term preterm infants.

[0018] In the above applications, the reagents required for detecting the relative expression levels of APOE and CST3 in placental RNA using real-time quantitative PCR can be the same reagents required for detecting the relative expression levels of APOE and CST3 in placental RNA relative to the internal control using real-time quantitative PCR. The internal control can be 18S rRNA.

[0019] In the above applications, the reagents required for detecting the expression levels of APOE and CST3 relative to the internal reference in placental RNA using the real-time quantitative PCR method may include primer pairs for detecting APOE, primer pairs for detecting CST3, and primer pairs for detecting 18S rRNA.

[0020] In the above applications, the reagents required for detecting the expression levels of APOE and CST3 relative to the internal reference in placental RNA using the real-time quantitative PCR method can specifically consist of primer pairs for detecting APOE, primer pairs for detecting CST3, and primer pairs for detecting 18S rRNA.

[0021] The primer pair for detecting APOE can consist of a primer with a nucleotide sequence as shown in SEQ ID NO:1 and a primer with a nucleotide sequence as shown in SEQ ID NO:2.

[0022] The primer pair for detecting CST3 can consist of a primer with a nucleotide sequence as shown in SEQ ID NO:3 and a primer with a nucleotide sequence as shown in SEQ ID NO:4.

[0023] Primer pairs for detecting 18S rRNA can consist of primers with nucleotide sequences as shown in SEQ ID NO:5 and primers with nucleotide sequences as shown in SEQ ID NO:6.

[0024] This invention also protects the use of placental APOE and CST3 as biomarkers in the preparation of products for early screening of neurodevelopmental delay in mid-to-late-term preterm infants.

[0025] In the above applications, APOE and CST3 in placental RNA are used as biomarkers. The -ΔCt value is obtained based on the expression levels of APOE and CST3 in placental RNA. A machine learning model is trained to obtain a random forest machine learning model as a classification model (i.e., the "2-mRNA Panel" classification model). The classification model outputs a risk score between 0 and 1, and the risk score is used to determine whether neurodevelopmental delay is present. In the embodiments of this invention, when the risk score output by the "2-mRNA Panel" classification model is above 0.51, the risk of mid-to-late-term preterm infants having or suspected of having gross motor delay is relatively high; when the risk score output by the "2-mRNA Panel" classification model is less than 0.51, the risk of mid-to-late-term preterm infants having or suspected of having gross motor delay is relatively low. In the embodiments of this invention, when the risk score output by the "2-mRNA Panel" classification model is above 0.73, the risk of mid-to-late-term preterm infants having or suspected of having fine motor delay is relatively high. When the risk score output by the "Panel" classification model is less than 0.73, the risk of mid-to-late preterm infants having or being suspected of having fine motor delay is relatively low. In the embodiments of the present invention, when the risk score output by the "2-mRNA Panel" classification model is greater than 0.56, the risk of mid-to-late preterm infants having or being suspected of having personal and social delay is relatively high; when the risk score output by the "2-mRNA Panel" classification model is less than 0.56, the risk of mid-to-late preterm infants having or being suspected of having personal and social delay is relatively low.

[0026] In any of the above-described applications, the neurodevelopmental delay may be gross motor delay, fine motor delay, or personal-social delay.

[0027] In any of the above-described applications, the neurodevelopmental delay may be a 6-month-old neurodevelopmental delay.

[0028] The GenBank number of any of the aforementioned APOEs is 348. The GenBank number of any of the aforementioned CST3s is 1471.

[0029] This invention establishes a "2-mRNA Panel" classification model for early screening of neurodevelopmental delay in mid-to-late-term preterm infants based on the expression levels of two mRNAs, APOE and CST3, in the placenta. The area under the receiver operating characteristic curve (AUC) for gross motor delay, fine motor delay, and personal-social delay reached 86.1%, 82.2%, and 83.3%, respectively. For APOE and CST3 in the "2-mRNA Panel" classification model, the inventors validated the model using reverse transcription polymerase chain reaction in placental samples from independent cohorts. Differential expression analysis verified that, compared to neurodevelopmentally normal mid-to-late-term preterm infants, the expression of APOE and CST3 in placental RNA was significantly different in mid-to-late-term preterm infants with gross motor delay, fine motor delay, and personal-social delay. Therefore, the "2-mRNA Panel" classification model provided by this invention uses the placenta as the detection target. By utilizing the expression levels of two mRNAs, APOE and CST3, in the placenta, early screening for neurodevelopmental delay in mid-to-late-term preterm infants can be performed. Thus, APOE and CST3 mRNAs in the placenta can serve as biomarkers for neurodevelopmental delay in mid-to-late-term preterm infants, providing a valuable supplement to the development of early screening tools for neurodevelopmental delay. This invention has significant application value. Attached Figure Description

[0030] Figure 1 ROC curve analysis results for predicting gross motor retardation in mid-to-late preterm infants using the "2-mRNA Panel" classification model.

[0031] Figure 2 ROC curve analysis results for predicting fine motor delay in mid-to-late preterm infants using the "2-mRNA Panel" classification model.

[0032] Figure 3 ROC curve analysis results for predicting personal-social functional delays in mid-to-late preterm infants using the "2-mRNA Panel" classification model.

[0033] Figure 4 To validate the biomarkers APOE and CST3 in the “2-mRNA Panel” classification model using reverse transcription polymerase chain reaction, the validation cohort consisted of four 6-month-old mid-to-late preterm infants with gross motor delay and nine 6-month-old mid-to-late preterm infants with normal neurodevelopment. Mann-Whitney U test was used. * indicates statistical significance, p < 0.05.

[0034] Figure 5To validate the biomarkers APOE and CST3 in the “2-mRNA Panel” classification model using reverse transcription polymerase chain reaction, the validation cohort consisted of 5 mid-to-late preterm infants with fine motor delay at 6 months of age and 9 mid-to-late preterm infants with normal neurodevelopment at 6 months of age. Mann-Whitney U test was used. * indicates statistical significance, p < 0.05.

[0035] Figure 6 To validate the biomarkers APOE and CST3 in the “2-mRNA Panel” classification model using reverse transcription polymerase chain reaction, the validation cohort consisted of 7 six-month-old mid-to-late preterm infants with personal-social delays and 9 six-month-old mid-to-late preterm infants with normal neurodevelopment. Mann-Whitney U test was used. ** indicates statistical significance, p < 0.01. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0039] In the following examples, unless otherwise specified, the first position of each nucleotide sequence is a 5′ terminal nucleotide, and the last position is a 3′ terminal nucleotide.

[0040] In the following examples, all mothers of mid-to-late preterm infants (i.e., the placental providers for mid-to-late preterm infants) gave informed consent.

[0041] Sensitivity (true positive rate): The percentage of individuals who actually have the disease but are correctly diagnosed according to the test criteria. Higher sensitivity is better; ideally, sensitivity is 100%.

[0042] Specificity (true negative rate): The percentage of individuals who are actually disease-free but are correctly diagnosed as disease-free according to the test criteria. Higher specificity is better; ideal specificity is 100%.

[0043] Mid-to-late term premature infants refer to live births between 32 and 37 weeks of gestation.

[0044] Example

[0045] I. Research Subjects

[0046] Neurodevelopmental Delay Group in Mid-to-Late Preterm Infants: The neurodevelopment of mid-to-late preterm infants at 6 months of age was assessed using the Ages and Stages Questionnaire of China (ASQ-C). Inclusion criteria were placentas of mid-to-late preterm infants whose scores in the gross motor skills area, fine motor skills area, or personal-social skills area were below one standard deviation of the norm mean (mean of standardized samples). The neurodevelopmental delay group consisted of placentas from 16 mid-to-late preterm infants with neurodevelopmental delay, encompassing gross motor delay, fine motor delay, and personal-social skills delay.

[0047] Neurodevelopmentally normal group of mid-to-late-term preterm infants: The neurodevelopment of 6-month-old mid-to-late-term preterm infants was assessed using the Ages and Stages Questionnaire of China (ASQ-C). Inclusion criteria were placentas from mid-to-late-term preterm infants whose scores in all five functional areas (gross motor skills, fine motor skills, communication skills, problem-solving skills, and personal-social skills) were higher than the norm mean by one standard deviation. The neurodevelopmentally normal group of mid-to-late-term preterm infants consisted of placentas from 9 neurodevelopmentally normal mid-to-late-term preterm infants.

[0048] II. Reverse transcription polymerase chain reaction (RT-PCR) detection of 2-mRNA in placental total RNA. The -ΔCt values ​​of APOE and CST3 in the panel (composed of APOE and CST3) were analyzed.

[0049] 1. Total RNA was extracted from the placenta using TRIzol (a product of Invitrogen, catalog number 15596-026).

[0050] 2. Total RNA was collected from the placenta and reverse transcribed using the Evo M-MLV cDNA First-Strand Synthesis Kit (product of Aikerui Biotechnology Co., Ltd., product catalog number AG11707) to obtain placental cDNA.

[0051] The reaction system consisted of 10 μl of placental cDNA (approximately 599 ng), 2 μl of 5×Evo M-MLV RT Master Mix, and RNase-free water. Both the 5×Evo M-MLV RT Master Mix and RNase-free water are components of the Evo M-MLV cDNA first-strand synthesis kit.

[0052] The reaction procedure was: 37℃ for 15 min, 85℃ for 5 sec.

[0053] 3. Using placental cDNA as a template, qPCR Green Master Mix (No Rox) (YESEN product, catalog number 11201ES08) was used for reverse transcription polymerase chain reaction in a real-time PCR instrument. Based on the output Ct value, 2... -ΔCt The -ΔCt values ​​of APOE and CST3 were obtained by means of 18S rRNA as an internal reference.

[0054] The reaction system consisted of 20 μl of placental cDNA, 0.4 μl of forward primer aqueous solution (10 μM), 0.4 μl of reverse primer aqueous solution (10 μM), and 10 μl of... qPCR It consists of Green Master Mix (No Rox) and nuclease-free water.

[0055] Reaction program: 95℃ for 5 min; 95℃ for 10 s, 60℃ for 30 s, 40 cycles.

[0056] The nucleotide sequences of the forward and reverse primers for detecting APOE, CST3, and 18S rRNA are shown in Table 1.

[0057] Table 1

[0058]

[0059] III. Constructing the “2-mRNA Panel” classification model

[0060] The inventors of this invention trained a machine learning model based on the -ΔCt values ​​of APOE and CST3 reverse transcription polymerase chain reactions, ultimately determining a random forest machine learning model as the classification model (i.e., the "2-mRNA Panel" classification model). The training data consisted of the -ΔCt values ​​of the reverse transcription polymerase chain reactions, with APOE and CST3 as the two biomarkers. To determine the hyperparameters of the random forest, the inventors optimized it using a grid search algorithm (using the GridSearchCV class from the scikit-learn Python package). The number of decision trees (the parameter n_estimators in the sklearn.ensemble.randomforest classifier) ​​could be 25, 50, or 75; the maximum tree depth (the parameter max_depth) could be 3, 4, or 5. The final random forest model for classifying neurodevelopmental delay was obtained, with the specific parameters being RandomForestClassifier(bootstrap=True, class_weight=None, criterion='gini', max_depth=5, max_features='auto', max_leaf_nodes=None, min_impurity_decrease=0.0, min_impurity_split=None, min_samples_leaf=1, min_samples_split=2, min_weight_fraction_leaf=0.0, n_estimators=25, n_jobs=None, oob_score=False, random_state=None, verbose=0, warm_start=False). The inventors of this invention saved the classification model as a pkl file. The model output is a risk score between 0 and 1. Neurodevelopmental delay is determined based on the risk score and the following criteria:

[0061] When the risk score output by the "2-mRNA Panel" classification model is above 0.51, the risk of mid-to-late-term preterm infants having or being suspected of having gross motor retardation is relatively high; when the risk score output by the "2-mRNA Panel" classification model is less than 0.51, the risk of mid-to-late-term preterm infants having or being suspected of having gross motor retardation is relatively low.

[0062] When the risk score output by the "2-mRNA Panel" classification model is above 0.73, the risk of mid-to-late-term preterm infants having or being suspected of having fine motor delay is relatively high; when the risk score output by the "2-mRNA Panel" classification model is less than 0.73, the risk of mid-to-late-term preterm infants having or being suspected of having fine motor delay is relatively low.

[0063] When the risk score output by the "2-mRNA Panel" classification model is above 0.56, the risk of mid-to-late-term preterm infants having or being suspected of having personal and social dysfunction is relatively high; when the risk score output by the "2-mRNA Panel" classification model is less than 0.56, the risk of mid-to-late-term preterm infants having or being suspected of having personal and social dysfunction is relatively low.

[0064] The 2-mRNA Panel consists of APOE and CST3, and the classification model is a random forest model.

[0065] The ROC curve analysis results of the “2-mRNA Panel” classification model for predicting gross motor delay in mid-to-late preterm infants are shown in [the table]. Figure 1 The AUC reaches 86.1%. The model has a specificity of 75% and a sensitivity of 89%.

[0066] The ROC curve analysis results of the “2-mRNA Panel” classification model for predicting fine motor delay in mid-to-late preterm infants are shown in [the table]. Figure 2 The AUC reaches 82.2%. The model has a specificity of 60% and a sensitivity of 78%.

[0067] The ROC curve analysis results of the “2-mRNA Panel” classification model predicting personal-social dysfunction in mid-to-late preterm infants are shown in [the table]. Figure 3 The AUC reaches 83.3%. The model's specificity is 71%, and its sensitivity is 78%.

[0068] IV. Clinical Validation Results

[0069] 1. Slow gross motor skills

[0070] (1) For APOE and CST3 in the “2-mRNA Panel” classification model, the inventors of this invention used reverse transcription polymerase chain reaction to verify them in placental samples from the normal group and the gross motor dyskinetic group.

[0071] Normal group: The neurodevelopment of mid-to-late-term preterm infants at 6 months of age was assessed using the Ages and Stages Questionnaire of China (ASQ-C). Inclusion criteria were placentas from mid-to-late-term preterm infants whose scores in all five functional areas (gross motor skills, fine motor skills, communication skills, problem-solving skills, and personal-social skills) were higher than the norm mean by one standard deviation. The normal group consisted of placentas from 9 mid-to-late-term preterm infants with normal neurodevelopment.

[0072] Gross motor dementia group: Neurodevelopment of mid-to-late-term preterm infants at 6 months of age was assessed using the Ages and Stages Questionnaire of China (ASQ-C). Inclusion criteria were placentas from mid-to-late-term preterm infants whose gross motor function scores were below the norm mean by one standard deviation. The gross motor dementia group consisted of placentas from four mid-to-late-term preterm infants with gross motor dementia.

[0073] (2) Differential expression analysis confirmed that, compared with neurodevelopmentally normal mid-to-late-term preterm infants, the expression of APOE and CST3 in placental RNA of mid-to-late-term preterm infants with gross motor delay was significantly different (see [link to study]). Figure 4 (Normal group is defined as normal, and abnormal group is defined as gross motor dysfunction).

[0074] 2. Fine motor skill delay

[0075] (1) For APOE and CST3 in the “2-mRNA Panel” classification model, the inventors of this invention verified them using reverse transcription polymerase chain reaction in placental samples from the normal group and the fine motor dyskinia group.

[0076] Normal group: The neurodevelopment of mid-to-late-term preterm infants at 6 months of age was assessed using the Ages and Stages Questionnaire of China (ASQ-C). Inclusion criteria were placentas from mid-to-late-term preterm infants whose scores in all five functional areas (gross motor skills, fine motor skills, communication skills, problem-solving skills, and personal-social skills) were higher than the norm mean by one standard deviation. The normal group consisted of placentas from 9 mid-to-late-term preterm infants with normal neurodevelopment.

[0077] Fine motor development disorder group: The neurodevelopment of mid-to-late preterm infants at 6 months of age was assessed using the Ages and Stages Questionnaire of China (ASQ-C). Inclusion criteria were placentas of mid-to-late preterm infants whose fine motor function scores were below the norm mean by one standard deviation. The fine motor function disorder group consisted of placentas from 5 mid-to-late preterm infants with fine motor development disorder.

[0078] (2) Differential expression analysis confirmed that, compared with neurodevelopmentally normal mid-to-late preterm infants, the expression of APOE and CST3 in placental RNA of mid-to-late preterm infants with fine motor delay was significantly different (see [link to study]). Figure 5 (Normal group is defined as normal, and abnormal group is defined as fine motor slowness abnormality).

[0079] 3. Delayed personal-social functioning

[0080] (1) For APOE and CST3 in the “2-mRNA Panel” classification model, the inventors of this invention verified them using reverse transcription polymerase chain reaction in placental samples from the normal group and the individual-social dysfunction group.

[0081] Normal group: The neurodevelopment of mid-to-late-term preterm infants at 6 months of age was assessed using the Ages and Stages Questionnaire of China (ASQ-C). Inclusion criteria were placentas from mid-to-late-term preterm infants whose scores in all five functional areas (gross motor skills, fine motor skills, communication skills, problem-solving skills, and personal-social functioning) were above the norm mean by one standard deviation. The normal group consisted of placentas from 9 mid-to-late-term preterm infants with normal neurodevelopment.

[0082] The Personal-Social Delay Group: Neurodevelopment of mid-to-late-term preterm infants at 6 months of age was assessed using the Ages and Stages Questionnaire of China (ASQ-C). Inclusion criteria were placentas from mid-to-late-term preterm infants whose Personal-Social Delay scores were below the norm mean by one standard deviation. The Personal-Social Delay Group consisted of placentas from seven mid-to-late-term preterm infants with Personal-Social Delay.

[0083] (2) Differential expression analysis confirmed that, compared with neurodevelopmentally normal mid-to-late preterm infants, the expression of APOE and CST3 in placental RNA of mid-to-late preterm infants with personal-social dysfunction was significantly different (see [link to study]). Figure 6 (Normal group is defined as normal, and abnormal group is defined as individual-social dysfunction).

[0084] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of reagents required for detecting relative expression of APOE and CST3 in placental RNA by real-time fluorescent quantitative PCR in the preparation of a product for early screening of neurodevelopmental retardation in moderate and late preterm infants. The neurodevelopmental retardation is gross motor retardation, fine motor retardation or personal-social function retardation. The neurodevelopmental retardation is neurodevelopmental retardation at 6 months of age.

2. Use according to claim 1, characterized in that: The reagents required for detecting relative expression of APOE and CST3 in placental RNA by real-time fluorescent quantitative PCR are reagents required for detecting relative expression of APOE and CST3 in placental RNA by real-time fluorescent quantitative PCR; the internal reference is 18S rRNA.

3. Use according to claim 2, characterized in that: The reagents required for detecting relative expression of APOE and CST3 in placental RNA by real-time fluorescent quantitative PCR comprise a primer pair for detecting APOE, a primer pair for detecting CST3 and a primer pair for detecting 18S rRNA. The primer pair for detecting APOE consists of a primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a primer with a nucleotide sequence as shown in SEQ ID NO:

2. The primer pair for detecting CST3 consists of a primer with a nucleotide sequence as shown in SEQ ID NO: 3 and a primer with a nucleotide sequence as shown in SEQ ID NO:

4. The primer pair for detecting 18S rRNA consists of a primer with a nucleotide sequence as shown in SEQ ID NO: 5 and a primer with a nucleotide sequence as shown in SEQ ID NO: 6.

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