CN110863066A - Kit for detecting five TORCH pathogens and application thereof - Google Patents
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a primer probe combination for detecting five TORCH pathogens, which comprises 5 pairs of primers and 5 corresponding probes. The invention also provides a kit for detecting five TORCH pathogens, which comprises a primer probe combination for detecting five TORCH pathogens. The invention also provides application of the kit for detecting five TORCH pathogens in non-diagnostic purposes in detection or auxiliary detection of the five TORCH pathogens. The kit for detecting five TORCH pathogens provided by the invention can be used for simultaneously detecting five TORCH pathogens, realizes multiple detection, has high sensitivity, is quick and convenient, and can realize batch detection of samples.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a kit for detecting five TORCH pathogens and application thereof.
Background
TORCH is a group of pathogens that can cause congenital intrauterine and perinatal infections, including toxoplasma gondii (TOX), Rubella Virus (RV), human Cytomegalovirus (CMV), herpes simplex virus type I (HSV1), and herpes simplex virus type II (HSV 2).
After a fertile woman becomes infected with one or more of the pathogens of TORCH, the pathogens are transmitted vertically through the placenta to the fetus, which can lead to abortion, premature birth, dead fetus, and malformation. Fetal deformity caused by toxoplasma includes hydrocephalus, cerebellar deformity, chorioretinitis and encephalalcification, congenital cataract, congenital heart disease and nerve deafness of fetus caused by rubella virus infection, growth retardation, small head, encephalitis, retinal vasculitis, jaundice, hepatosplenomegaly, hemolytic anemia and the like of fetus caused by cytomegalovirus infection, and abortion or neonatal morbidity caused by herpes simplex virus I/II infection.
At present, the clinical detection of the TORCH pathogen is mainly carried out by adopting an immunological method, such as an ELISA (enzyme-linked immunosorbent assay) or a colloidal gold method, and detecting IgG or IgM (immunoglobulin M) generated after a human body is infected with the pathogen, wherein the IgM is recently infected or a latent virus is activated to generate recurrent infection, and the IgG is the previous infection and has a certain immunity level. However, the immunological method for detecting the above 5 pathogens is not only poor in sensitivity and specificity, but also may cause false negative in a certain window period after the infection until IgG or IgM is produced, and is not favorable for the patient to know the detection result in time due to the complex reagent and long operation time.
In recent years, with the development of molecular biology, nucleic acid detection methods have the characteristics of high speed, high sensitivity and good specificity, and have been widely used for clinical detection. In view of the above, a need exists for a nucleic acid detection method for detecting five pathogens of TORCH, which has high sensitivity, good specificity, simple experimental operation, and short time consumption.
Disclosure of Invention
The invention aims to provide a primer probe combination for detecting five TORCH pathogens.
The invention also aims to provide a kit for detecting five TORCH pathogens and application thereof, the kit can be used for simultaneously detecting the five TORCH pathogens, multiple detection is realized, the sensitivity is high, the kit is quick and convenient, and batch detection of samples can be realized.
To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a primer-probe combination for detecting five pathogens of TORCH, characterized in that the primer-probe combination includes:
1) a primer pair 1 with polynucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 and a probe 1 with polynucleotide sequences shown as SEQ ID NO. 11;
2) a primer pair 2 with polynucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO.4 and a probe 2 with polynucleotide sequences shown as SEQ ID NO. 12;
3) a primer pair 3 sequence shown by polynucleotide sequences as SEQ ID NO.5 and SEQ ID NO.6 and a probe 3 shown by polynucleotide sequence as SEQ ID NO. 13;
4) a primer pair 4 sequence shown by polynucleotide sequences as SEQ ID NO.7 and SEQ ID NO.8 and a probe 4 shown by polynucleotide sequence as SEQ ID NO. 14;
5) a primer pair 5 sequence shown by polynucleotide sequences as SEQ ID NO.9 and SEQ ID NO.10 and a probe 5 shown by polynucleotide sequence as SEQ ID NO. 15.
Wherein the content of the first and second substances,
the single-stranded DNA probe 1 is used for monitoring the amplification result of the primer pair 1 in real time;
the single-stranded DNA probe 2 is used for monitoring the amplification result of the primer pair 2 in real time;
the single-stranded DNA probe 3 is used for monitoring the amplification result of the primer pair 3 in real time;
the single-stranded DNA probe 4 is used for monitoring the amplification result of the primer pair 4 in real time;
the single-stranded DNA probe 5 is used for monitoring the amplification result of the primer pair 5 in real time;
preferably, the 5' end of the probe is labeled with any one of fluorescent reporter groups, FAM, JOE, ROX or Cy 5; the 3' end of the probe is marked with a fluorescence quenching group BHQ 1.
The kit for detecting five TORCH pathogens comprises the primer probe combination for detecting five TORCH pathogens.
Preferably, the kit further comprises PCR buffer, DNA polymerase, reverse transcriptase, an internal control and a positive control, wherein the internal control is human β -globin, and the positive control is a plasmid containing amplification sequences of five TORCH pathogens.
Preferably, the five TORCH pathogens are toxoplasma TOX, rubella virus RV, human cytomegalovirus CMV, herpes simplex virus I HSV1 and herpes simplex virus II HSV 2.
The application of the kit for detecting five TORCH pathogens in the detection or auxiliary detection of five TORCH pathogens for non-diagnostic purposes.
Preferably, the reaction system of the kit in detecting or assisting in detecting five pathogens of TORCH is 20uL, specifically:
2uL of nucleic acid template, 10uL of 2 XPCR buffer, 0.5uL of DNA polymerase, 0.5uL of reverse transcriptase, 2uL of mixed solution containing the primer and probe combination and 20uL of the rest DEPC-supplemented water;
wherein the final concentration of the primer pair in the amplification system is 100-1000 nM.
The invention has the beneficial effects that:
in conclusion, the kit for detecting five TORCH pathogens provided by the invention solves the problem of simultaneous detection and identification of five TORCH pathogens, can simultaneously detect and identify five TORCH pathogens, and has the advantages of simple and convenient operation, accurate and sensitive detection, good specificity and rapidness.
The kit for detecting five TORCH pathogens provided by the invention can realize multiple detections on samples and can perform batch detection on the samples.
Drawings
FIG. 1 is a sensitivity test of a primer probe set, in which ten-fold gradient dilution is performed for home-made TOX pathogen detection positive plasmid samplesRear (10)2copies/ul、103copies/ul、104copies/ul and 105copies/ul) of the amplification reaction and the resulting amplification curve, R2A value of 0.999;
FIG. 2 is a sensitivity test of primer probe sets, wherein each sample is subjected to ten-fold gradient dilution (10) for home-made RV pathogen detection positive plasmid samples2copies/ul、103copies/ul、104copies/ul and 105copies/ul), the amplification curve obtained by carrying out the amplification reaction, R2A value of 0.996;
FIG. 3 shows the sensitivity test of the primer probe set, wherein the CMV pathogen detection positive plasmid samples prepared by self-made were diluted in ten-fold gradient (10)2copies/ul、103copies/ul、104copies/ul and 105copies/ul) of the amplification reaction and the resulting amplification curve, R2A value of 0.998;
FIG. 4 shows the sensitivity test of primer probe set, wherein each sample is a ten-fold gradient dilution of self-made HSV1 pathogen detection positive plasmid (10)2copies/ul、103copies/ul、104copies/ul and 105copies/ul) of the amplification reaction and the resulting amplification curve, R2A value of 0.998;
FIG. 5 shows the sensitivity test of primer probe set, wherein each sample is a ten-fold gradient dilution of self-made HSV2 pathogen detection positive plasmid (10)2copies/ul、103copies/ul、104copies/ul and 105copies/ul) of the amplification reaction and the resulting amplification curve, R2The value was 0.991.
Detailed Description
The present invention is described in further detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
Detection of five pathogens of TORCH:
(1) extraction of genomic DNA
A sample of a person to be detected is collected by adopting a commercially available virus genome DNA/RNA extraction kit, and the specific operation process is carried out according to the instruction.
(2) Primer design
Corresponding sequences of toxoplasma gondii (TOX), Rubella Virus (RV), human Cytomegalovirus (CMV), herpes simplex virus I (HSV1) and herpes simplex virus II (HSV2) are obtained from GenBank, and amplification primer probes are designed to ensure that each pair of primer probes can amplify corresponding pathogens and do not carry out non-specific amplification with other pathogens.
TABLE 1 TORCH five pathogen amplification primers and probes
(3) Establishment of multiplex PCR method
By using the kit provided by the application, the reaction system is 20uL, specifically: 2uL of nucleic acid template, 10uL of 2 XPCR buffer, 0.5uL of DNA polymerase, 0.5uL of reverse transcriptase, 2uL of primer probe mixture and 20uL of DEPC water. The nucleic acid amplification reaction solution contains magnesium chloride (25mM), 10mM dNTP, 200mM KCl and 20mM tetramethylammonium chloride, the final concentration of the primer pair in the system is 500nM, and the final concentration of the probe in the system is 250 nM. Putting the mixture into a fluorescent quantitative PCR instrument, setting a reaction program, and starting detection under the following reaction conditions: 50 ℃ for 10 min; 95 ℃ for 5 min; [95 ℃, 15 s; 60 ℃, 55s ]40 cycles. The detection probe channel and grouping are shown in table 2.
TABLE 2 detection Probe channel and grouping
(4) Results
After the reaction is finished, analyzing the detection result according to the amplification curve graph and the CT value:
the fluorescence curve in the FAM channel is an S-shaped curve, CT is less than or equal to 37.5, and the fluorescence curve is judged to be positive; no typical "S" type amplification or CT >37.5, and CT of internal control is less than or equal to 37.5, and the result is judged to be negative.
The fluorescence curve in the CY5 channel is S-shaped curve and CT is less than or equal to 38, and the result is judged to be positive; no typical "S" type amplification or CT >38, and CT of internal control is less than or equal to 38, and the result is judged to be negative.
The fluorescence curve in the JOE channel is an S-shaped curve, CT is less than or equal to 38.5, and the JOE channel is judged to be positive; no typical "S" type amplification or CT >38.5, and the internal control CT < 38.5, judged negative.
The fluorescence curve in the ROX channel is an S-shaped curve, CT is less than or equal to 38, and the ROX channel is judged to be positive; no typical "S" type amplification or CT >38, and CT of internal control is less than or equal to 38, and the result is judged to be negative.
Example 2
Specificity test of primer Probe set
Five pathogen plasmids of TORCH were used to cross-test with known pathogen samples to verify their specificity.
The corresponding primer probes of 5 TORCH pathogens are added into the wells of the same row of a 96-well plate, the positive plasmid templates of the same 5 TORCH pathogens and samples of known pathogens are added into the wells of the same row, the samples of the known pathogens comprise influenza virus A (FluA), influenza virus B (FluB), coronavirus (OC43), bocavirus (HBOV), parainfluenza virus 1(HPIV1), parainfluenza virus 3(HPIV3), rhinovirus (HRV), Mycoplasma Pneumoniae (MP) and Chlamydia Pneumoniae (CP), each primer probe and any pathogen are ensured to have independent contact opportunities, and the specificity of the method is verified by counting CT values through cross experiments.
The multiplex fluorescence PCR reaction system is 20uL, and specifically comprises: each group of pathogens mixes 2uL of DNA sample, 10uL of 2 XPCR buffer, 0.5uL of DNA polymerase, 0.5uL of reverse transcriptase, 2uL of primer probe mixture, and 20uL of DEPC-supplemented water. Putting the mixture into a fluorescent quantitative PCR instrument, setting a reaction program, and starting detection under the following reaction conditions: 50 ℃ for 10 min; at 95 ℃ for 3 min; [95 ℃, 5 s; 60 ℃, 45s ]40 cycles. The results are shown in Table 3, and it can be seen from the results in Table 3 that the primer probes in each group only amplified with the corresponding pathogen, indicating that each primer probe has good specificity.
TABLE 3 results of experiments on the specificity of primer probe sets for the method of the invention
| TOX | RV | CMV | HSV1 | HSV2 | |
| TOX | + | - | - | - | - |
| RV | - | + | - | - | - |
| CMV | - | - | + | - | - |
| HSV1 | - | - | - | + | - |
| HSV2 | - | - | - | - | + |
| FluA | - | - | - | - | - |
| FluB | - | - | - | - | - |
| OC43 | - | - | - | - | - |
| HBOV | - | - | - | - | - |
| HPIV1 | - | - | - | - | - |
| HPIV3 | - | - | - | - | - |
| HRV | - | - | - | - | - |
| MP | - | - | - | - | - |
| CP | - | - | - | - | - |
Example 3
Sensitivity test
Ten-fold dilution of 5 self-made TORCH pathogen positive plasmid samples, 102-105copy/ul, the diluted samples were amplified separately using the kit provided in this patent, and the results of the sensitivity of detection of 5 TORCH pathogens are shown in FIGS. 1-5, and their R is2The values are 0.999(TOX), 0.996(RV), 0.998(CMV), 0.998 (HSV1) and 0.991(HSV2) respectively, which shows that the linear relation is good, and the sensitivity can reach 100 copies/ul.
Example 4
Accuracy test
10 samples of the TORCH pathogens are randomly selected, the kit is used for detection, sequences obtained by sample sequencing are subjected to Blast comparison, and the coincidence rate of the detection result and the sequencing result of the kit is 100%.
Example 5
Repeatability test
Will 102-105Samples of 5 TORCH pathogen positive plasmids from copies/ul were plated in 3 wells in parallel and the coefficient of variation between CT values was calculated for 3 parallel samples. The results are shown in Table 4 below, and the coefficient of variation is below 4%, which shows that the kit of the invention has good repeatability.
TABLE 4 repetitive results of the kit of the present invention for detecting 5 TORCH pathogens
Example 6
Detection of actual clinical samples
The clinical samples used in this example are from blood/secretion samples collected by hospitals in a certain area of Hebei province (principle of volunteers), and total 500 cases. And (4) extracting nucleic acid. The kit is used for carrying out multiple fluorescence PCR detection, and the result (shown in the following table 5) is obtained by comparing the kit with an internal reference substance and a positive reference substance.
TABLE 5 test results of the clinical specimens with the kit of the present invention
| Number of positive cases in the invention | Number of ELISA positives | |
| TOX | 0 | 0 |
| RV | 4 | 4 |
| CMV | 7 | 7 |
| HSV1 | 3 | 3 |
| |
5 | 5 |
While embodiments of the invention have been disclosed above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in a variety of fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
<110> Gallery norway Zhongke medical laboratory Co., Ltd
<120> kit for detecting TORCH pathogen and application thereof
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Claims (7)
1. A primer probe combination for detecting five TORCH pathogens, which is characterized in that the primer probe combination comprises:
1) a primer pair 1 with polynucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 and a probe 1 with polynucleotide sequences shown as SEQ ID NO. 11;
2) a primer pair 2 with polynucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO.4 and a probe 2 with polynucleotide sequences shown as SEQ ID NO. 12;
3) a primer pair 3 sequence shown by polynucleotide sequences as SEQ ID NO.5 and SEQ ID NO.6 and a probe 3 shown by polynucleotide sequence as SEQ ID NO. 13;
4) a primer pair 4 sequence shown by polynucleotide sequences as SEQ ID NO.7 and SEQ ID NO.8 and a probe 4 shown by polynucleotide sequence as SEQ ID NO. 14;
5) a primer pair 5 sequence shown by polynucleotide sequences as SEQ ID NO.9 and SEQ ID NO.10 and a probe 5 shown by polynucleotide sequence as SEQ ID NO. 15.
2. The primer-probe combination for detecting five TORCH pathogens according to claim 1, wherein the 5' end of the probe is labeled with any one of fluorescent reporter groups FAM, JOE, ROX or Cy 5; the 3' end of the probe is marked with a fluorescence quenching group BHQ 1.
3. Kit for the detection of five pathogens of TORCH, comprising the primer-probe combination according to any one of claims 1 or 2 for the detection of five pathogens of TORCH.
4. The kit for detecting five pathogens of TORCH of claim 3, further comprising PCR buffer, DNA polymerase, reverse transcriptase, an internal control, wherein the internal control is human β -globin, and a positive control, wherein the positive control is a plasmid comprising amplified sequences of five pathogens of TORCH.
5. The kit for detecting five pathogens of TORCH of claim 4, wherein the five pathogens of TORCH are toxoplasma toxm, rubella virus RV, human cytomegalovirus CMV, herpes simplex virus type I HSV1, and herpes simplex virus type II HSV 2.
6. Use of the kit for detecting five pathogens of TORCH according to claim 3 for non-diagnostic purposes in the detection or assisted detection of five pathogens of TORCH.
7. The use according to claim 6, wherein the reaction system of the kit in detecting or assisting in detecting five pathogens of TORCH is 20uL, specifically:
2uL of nucleic acid template, 10uL of 2 XPCR buffer, 0.5uL of DNA polymerase, 0.5uL of reverse transcriptase, 2uL of mixed solution containing the primer and probe combination and 20uL of the rest DEPC-supplemented water; wherein the final concentration of the primer probe combination in the amplification system is 100-1000 nM.
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Cited By (2)
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| CN112301169A (en) * | 2020-12-30 | 2021-02-02 | 爱科睿特生物医疗科技(南京)有限公司 | Primer group, probe group and kit for synchronously detecting pathogens related to multiple genital tract infections |
| CN114231649A (en) * | 2021-12-24 | 2022-03-25 | 廊坊诺道中科医学检验实验室有限公司 | Primer-probe combination for detecting five TORCH pathogens, kit and application thereof |
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| CN114231649A (en) * | 2021-12-24 | 2022-03-25 | 廊坊诺道中科医学检验实验室有限公司 | Primer-probe combination for detecting five TORCH pathogens, kit and application thereof |
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