NL2033870B1 - Kit for detection of mycoplasma pneumoniae, detection method and use thereof - Google Patents
Kit for detection of mycoplasma pneumoniae, detection method and use thereof Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention discloses a kit for detection of Mycoplasma pneumoniae, a detection method and use thereof. Specifically disclosed is a primer pair consisting of primer FH-F and primer FH-R, the primer FH-F can be the single-stranded DNA molecule shown in SEQ ID No. l, the primer FH-R can be the single-stranded DNA molecule shown in SEQ ID No.2. The present invention also discloses a composition for detecting Mycoplasma pneumoniae and a real-time fluorescent RPA detection method. The primer pair, composition and detection method of the present invention have the advantages of simplicity, rapidity, sensitivity and specificity, the lowest detection limit can reach 10 fg/uL, the specificity is good, and there is no cross-reaction with common respiratory pathogens and other Mycoplasma pathogens. It can be applied to the detection of Mycoplasma pneumoniae and the diagnosis, screening, prognosis assessment, therapeutic monitoring and other fields of diseases caused by Mycoplasma pneumoniae infection. It is suitable for rapid on-site detection and has extremely broad application prospect.
Description
KIT FOR DETECTION OF MYCOPLASMA PNEUMONIAE, DETECTION METHOD AND
USE THEREOF
[0001] The present invention belongs to the technical field of clinical detection, relates to a kit for detecting Mycoplasma pneumoniae, a detection method and use thereof, and in particular relates to a primer-probe composition, a kit, a detection method and use for the detection of
Mycoplasma pneumoniae by recombinase polymerase amplification combined with real-time fluorescent probe.
[0002] Mycoplasma pneumoniae (MP) 1s one of the most common pathogens of community- acquired pneumonia in children and adolescents. During the MP epidemic, community-acquired pneumonia caused by MP accounts for about 20-40%, and can be as high as 70% in densely populated places such as schools. The clinical manifestations of MP infection are diverse. Mild patients only show mild respiratory tract infection symptoms, and severe patients can present with severe pneumonia. About 25% of severe patients may be accompanied by severe extrapulmonary complications. The clinical manifestations of MP are nonspecific and are ineffective against conventional B-lactam antibiotics for respiratory infections. Therefore, the development of a simple, rapid and reliable diagnostic method for MP is crucial for the rational use of antibiotics and the effective treatment of MP infectious diseases.
[0003] At present, the commonly used methods for the laboratory diagnosis of MP infection mainly include culture method, serological detection and molecular diagnosis method. The culture method is the gold standard for MP diagnosis, but the MP genome is small, and the culture conditions and laboratory requirements are strict. MP grows slowly and has a long culture period, usually 7-10 days. It takes 3-4 weeks to make a judgment, and the detection rate is relatively low. Therefore, the culture method is of little significance for early clinical diagnosis and treatment, and this method is mostly used in retrospective studies. Serological detection is still the most commonly used method for the diagnosis of MP infection at home and abroad because of its convenience and speed, and plays a very important role in the diagnosis of MP infection. However, serological antibody detection has the problems of window period and persistence of antibodies as well as large differences in sensitivity and specificity between different detection kits, thus limiting its clinical use. With the rapid development of nucleic acid diagnostic technology, some PCR-based diagnostic techniques (such as ordinary PCR technology, fluorescent PCR technology) are used for the rapid diagnosis of MP, however, these
S methods rely on expensive equipment and require subsequent electrophoresis operations, expensive probe synthesis, and skilled operators. It is not possible for some backward laboratories, which limits the application of these technologies.
[0004] In order to overcome the disadvantages of PCR amplification technology, many isothermal amplification technologies have been developed in recent years. Compared with PCR technology, isothermal amplification technology does not rely on thermal cycling amplification equipment, and has fast reaction speed and good sensitivity. Therefore, isothermal amplification technology 1s conducive to the realization of rapid amplification, convenient detection and on- site diagnosis. Recombinase polymerase amplification (RPA) technology is a constant temperature amplification technology that relies on a variety of enzymes. The detection technology requires only 2 primers and can complete the amplification within 30 minutes, and has the advantages of high sensitivity and strong specificity. In addition, the detection technology 1s combined with real-time fluorescent probe detection technology, which can realize reliable reading of amplification products, eliminate aerosol pollution, and does not require complex instruments and equipment. It is suitable for rapid on-site detection and has extremely broad application prospects.
[0005] The technical problem to be solved by the present invention is how to detect
Mycoplasma pneumoniae (MP) simply, rapidly, accurately and/or reliably. The technical problem to be solved is not limited to the described technical subject, and other technical subjects not mentioned herein can be clearly understood by those skilled 1n the art from the following description.
[0006] In order to solve the aforementioned technical problems, the present invention first provides a reagent or kit for detecting Mycoplasma pneumoniae, the reagent or kit can comprise a composition for detecting Mycoplasma pneumoniae, the composition can include a primer pair and a probe, the primer pair can consist of primer FH-F and primer FH-R, the primer FH-F can be the single-stranded DNA molecule shown in SEQ ID No.1; and the primer FH-R can be the single-stranded DNA molecule shown in SEQ ID No.2.
[0007] The primer FH-F 18 a forward primer, and the primer FH-R is a reverse primer.
[0008] The forward primer FH-F and the reverse pruner FH-R are primer pairs for specifically amplifying Mycoplasma pneumoniae (MP), that is, a specific primer pair for detecting
Mycoplasma pneumoniae,
[0009] Further, in the aforementioned reagent or kit, the structure of the probe can be nucleotide fragment 1-tetrahydrofuran-nucleotide fragment 2, wherein the nucleotide sequence of nucleotide fragment 1 can be SEQ ID No. 3, and the nucleotide sequence of nucleotide fragment 2 can be SEQ ID No. 4.
[0010] Further, the 3' end of the nucleotide fragment 1 can be labeled with a fluorophore, and the 5' end of the nucleotide fragment 2 can be labeled with a quenching group.
[0011] The fluorophore is selected from at least one of FAM, 6-FAM, VIC, HEX, TRT, CY3,
CY5 ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705 or Texas
Red
[0012] The quenching group is selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3,
MGB, and Dabceyl.
[0015] Further, the fluorophore can be 6-FAM, and the quenching group can be BHQ1.
[0014] Further, the sequence of the probe is as follows:
ATACCAAGAGTGGTTCACAACACGATT/I6FAMAT/idsp/Tbhql d/ ATGTATGTCCT
TTG.
[0015] The probe contains tetrahydrofuran (THF), and THF 18 flanked by nucleotide fragment 1 and nucleotide fragment 2, respectively.
[0016] The nucleotide sequence of nucleotide fragment 1 is shown as below: 5 -ATACCAAGAGTGGTTCACAACACGATTT-6-FAM-3 (SEQ ID No.3) .
[0017] The nucleotide sequence of nucleotide fragment 2 is shown as below: 5’-BHQ1-TATGTATGTCCTTTG-3’ (SEQID No.4) .
Wherein, 1I0FAMAT is thymine T labeled with fluorophore 6-FAM (6- carboxytluorescein).
[0018] Idsp is abasic (base deletion) Tetrahydrofuran(THF).
[0019] Ibhqldt is a thymine T labeled with the quenching group BHT.
[0020] Further, the kit also includes one or more of a negative control, a positive control, and a buffer. The negative control can be a reaction system without the genome template of
Mycoplasma pneumoniae. [00211 The positive control can be a reaction system containing a genome template of
Mycoplasma pneumoniae (MP).
[0022] The various reagent components of the kit can be present in separate containers, or can be pre-combined in whole or in part into a reagent mixture.
[0023] Further, the kit can further include a readable carrier describing the method for detecting Mycoplasma pneumomae described herein. The readable carrier can be kit instructions for practicing the methods of the present invention {eg, instructions in printed form} or a computer-readable medium (eg, floppy disk, CD, etc.) on which information has been recorded.
[0024] The pruner pair {primer FH-F and primer FH-R) or any of the compositions described herein are within the scope of the present invention.
[0025] The present invention further provides use of the primer pair, and/or, any of the compositions herein in any one of the following:
Al} use in the detection of Mycoplasma pneumoniae or in the preparation of a product for the detection of Mycoplasma pneumoniae;
A2} use in identifying or assisting the identification of Mycoplasma pneumoniae or in the preparation of a product for identifying or assisting the identification of Mycoplasma pneumoniae;
A3) use in diagnosing or assisting in the diagnosis of diseases caused by Mycoplasma pneumoniae infection or in the preparation of a product for diagnosing or assisting in the diagnosis of diseases caused by Mycoplasma pneumoniae infection;
Ad) use in screening diseases caused by Mycoplasma pneumoniae infection or in the preparation of a product for screening diseases caused by Mycoplasma pneumoniae infection;
AS} use in the prognosis assessment of diseases caused by Mycoplasma pneumoniae infection or in the preparation of a product for the prognosis assessment of diseases caused by
S Mycoplasma pneumoniae infection;
A6) use in therapeutic monitoring of diseases caused by Mycoplasma pneumoniae infection or in the preparation of a product for therapeutic monitoring of diseases caused by
Mycoplasma pneumoniae infection.
[0026] The products described herein can be reagents, kits, chips or test strips.
[00627] The present invention further provides a method for detecting Mycoplasma pneumoniae, the method can include using the primer pair (primer FH-F and primer FH-R) or any of the compositions described herein to carry out a recombinase polymerase amplification reaction (RPA reaction) on the sample to be tested, and determine whether the sample to be tested contains Mycoplasma pneumoniae or whether it 1s Mycoplasma pneumoniae according to the amplification product.
[0028] Further, the RPA reaction can be a real-time fluorescence quantitative RPA reaction.
[0029] The method for detecting Mycoplasma pneumoniae can be a real-time fluorescent RPA detection method.
[0030] In the aforementioned method, the temperature of the recombinase polymerase amplification reaction can be 37-41°C.
[0031] In the aforementioned method, the temperature of the recombinase polymerase amplification reaction can be 39°C.
[0032] Further, in the aforementioned method, the recombinase polymerase amplification reaction system includes: buffer A, buffer B, forward primer FH-F, reverse primer FH-R, probe, and template.
[0033] Further, in the aforementioned reaction system, the molar ratio of primer FH-F and primer FH-R can be 1:1.
[0034] Further, in the aforementioned reaction system, the molar ratio of primer FH-F, primer
FH-R and probe can be 1: 1:1 4.
[0035] Further, the system of the recombinase polymerase amplification reaction can be 29.5 ul buffer A, 2.5 uL buffer B, 10 uM forward primer FH-F 2 pL, 10 pM reverse primer FH-R 2
HL, 10 uL pM probe 0.6 pL, template 1 pL (template concentration 210 fg/pL), add deionized water to 50 ul.
[0036] Further, the template concentration can be 10 fg/uL.
[0037] Buffer A and buffer B are used for RPA amplification and are purchased from Weifang
AMP-Future Biotech Co, Ltd, catalog number: WLE8202KIT.
[0038] Further, the conditions of the recombinase polymerase amplification reaction can be a constant temperature of 39°C, the fluorescence value of the FAM channel is collected every 30 5, for a total of 40 cycles; the reaction time is 20 min. [00391 Further, the sample to be tested can be a variety of body fluid samples (sputum, lavage fluid, gastric juice, pleural effusion, blood, etc}, tissue samples, environmental samples (such as air), clothing or towels or animal tissues and/or organs used as food, etc. [00401 In the aforementioned method, the method for determining whether the sample to be tested contains Mycoplasma pneumoniae or whether it is Mycoplasma pneumoniae according to the amplification product is shown as below: [00411 If the sample to be tested shows UNDET or no typical S-shaped amplification curve, the report is negative; if the sample to be tested shows a typical “S-shaped” amplification curve, the CT value of the FAM channel 1s 37-40, and it needs to be re-tested again, if the test result is still 37-40, it is reported as negative;
[0042] If the sample to be tested shows a typical “S-shaped” amplification curve, the CT value of the FAM channel shows <37, and the report is positive.
[0043] The purpose of aforementioned use and method can be disease diagnosis purpose, disease prognosis purpose and/or disease treatment purpose, their purpose can also be non- disease diagnosis purpose, non-disease prognosis purpose and non-disease treatment purpose; their direct purpose can be to obtain information on intermediate results of disease diagnosis results, disease prognosis results and/or disease treatment results, and their direct purpose can be non-disease diagnosis purpose, non-disease prognostic purpose and/or non-disease treatment purpose.
[0044] Recombinase polymerase amplification (RPA) is an efficient isothermal nucleic acid amplification technology. The technical principle is: the protein-DNA complex formed by the
S combination of the recombinase and the primer can search for the homologous sequence in the double-stranded DNA. Once the primers locate the homologous sequence, a strand-exchange reaction takes place and initiates DNA synthesis, exponentially amplifying the target region on the template. The whole process ts very fast, and generally detectable levels of amplification products can be obtained within ten minutes. Due to the particularity of its technical principle,
PCR primers are often not suitable for RPA. The key to establishing the best RPA system with high sensitivity is to design primers and probes with high amplification efficiency and strong specificity. After extensive and in-depth research, the inventors designed a set of RPA amplification primers and probes (namely primer FH-F, primer FH-R and probe) for the specific gene of Mycoplasma pneumoniae (MP), and a simple, rapid, accurate and reliable MP diagnosis method based on RPA was established. The method is simple, rapid, sensitive and specific. The lowest detection limit can reach 10 fg/uL, and the specificity is good. H has no cross-reaction with common respiratory pathogens and other Mycoplasma pathogens. It can be applied to the detection of Mycoplasma pneumoniae and the diagnosis, screening, prognosis assessment, therapeutic monitoring and other fields of diseases caused by Mycoplasma pneumoniae infection. It is suitable for rapid on-site detection and has extremely broad application prospects.
[0045] FIG. 1 is the test result graph of the optimum reaction temperature of the real-time fluorescent RPA detection method in Example 2. In FIG. 1, a is the amplification curve when the reaction temperature is 37°C. In FIG. 1, b is the amplification curve when the reaction temperature is 38°C. In FIG. 1, c 1s the amplification curve when the reaction temperature is 39°C. In FIG. 1, d is the amplification curve when the reaction temperature is 40°C. In FIG. 1, ¢ is the amplification curve when the reaction temperature is 41°C.
[0046] FIG. Zis a graph showing the sensitivity detection results of the real-time fluorescent
RPA detection method in Example 4. In FIG. 2, a is the genome template of Mycoplasma pneumoniae with 1 fg, IO fg, 100 fg, 1 pg, 10 pg, and 100 pg for sensitivity venfication respectively. In FIG. 2, b is the genome template of Mycoplasma pneumoniae with 1 fg and 10 fg for sensitivity verification respectively.
[0047] FIG 3 1s a graph showing the specificity detection and evaluation of the real-time fluorescent RPA detection method in Example 5.
[0048] The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.
[0049] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The materials, reagents, etc. used 10 the following examples can be obtained from commercial sources unless otherwise specified. Wherein, buffer A and buffer B used for RPA amplification were purchased from
Weitang AMP-Future Biotech Co, Ltd. (Cat No. WLES202KIT).
[0050] Mycoplasma pneumomae (MP) M129 in the following examples is described in the following documents: Zhao F, Lm 7, Gu Y, Yang Y, Xiao D, Tao X, Meng F, He L, Zhang I.
Detection of Mycoplasma pneumoniae by colorimetric loop-mediated isothermal amplification.
Acta Microbiol Immunol Hung. 2013 Mar, 060(1):1-9. dor: 10.1556/AMicr.60.2013.1.1. The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.
[0051] Example 1. Design of primers and probes
In this example, a set of RPA amplification primers was designed for the specific gene of
Mycoplasma pneumoniae (MP } to further develop and verify the feasibility, sensitivity, specificity and reliability of RPA real-time fluorescence amplification technology.
[0052] This gene is present in all MPs, and its conservation and specificity are good, which can distinguish MPs from other closely related species. RPA primers were designed using the primer design software Primer Explorer V4 {Eiken Chemical) (http;//primerexplorerjp/e/) and the primer design software Primer Premier 5.0, and the obtained specific primers were subjected to sequence alignment analysis in the NCBI database (http://blast .nchi.nim.nh. gov/Blast.cgi} to exclude possible non-specific matches between primers and sequences of other species, and the
S optimized RPA amplification primers were finally obtained. The designed primer sequences and probes were shown in Table 1.
[0053] Table I. Primer and probe sequence information of RPA real-time fluorescent amplification detection method
UN Amplified
Primer/Probe Sequence (57-37)
Fragment
TGACACCGCAAGACAGTGCAATAACTC
FH-F
AGT (SEQ ID No.1)
CTGAACATCAACAAAGAAGGTGCTAGC
FH-R | Co 179 bp
TGC (SEQ ID No.2) : ATACCAAGAGTGGTTCACAACACGATTA probe i oF AMdT/idsp//Tbhg ldVATGTATGTCCTTTG
[0054] wherein, 1I6FAMAT was thymine T labeled with fluorophore 6-FAM (6- carboxyfluorescein).
[9055] Idsp was abasic (base deletion) tetrahydrofuran (Tetrahydrofuran, THF).
[0056] Ibhqldt was a thymine T labeled with the quenching group BHO].
[0057] The probe contained tetrahydrofuran (THF), and THF was flanked by nucleotide fragment | and nucleotide fragment 2, respectively.
[0058] The nucleotide sequence of nucleotide fragment 1 was shown as below: 3 -ATACCAAGAGTGGTTCACAACACGATTT-6-FAM-3" (SEQ ID No.3)
[0059] The nucleotide sequence of nucleotide fragment 2 was shown as below: 5’-BHQI-TATGTATGTCCTTTG-3’ (SEQID No4)
[0060] Example 2, The optimum reaction temperature of real-time fluorescence RPA detection [00611 1. Extraction of genomic DNA from the sample to be tested
[0062] The sample to be tested in this example was: Mycoplasma pneumoniae (MP) M129.
[0063] The MP genomic DNA was extracted using a DNA extraction kit from Qiagen (QlAamp DNA mini kits; Qiagen, Hilden, Germany) according to the instructions. The concentration and purity of genomic DNA were determined using a UV spectrophotometer, and the extracted MP genomic DNA was serially diluted in TE buffer (from 100 pg/ul, 10 pg/uL, 10 fg/uL, 1 fg/uL to 0.1 fg/ul ). Various genomic DNAs were aliquoted in small quantities and stored at -20°C for later use. The serially diluted MP genomic DNA was used to explore the optimal temperature of the RPA real-time fluorescence amplification and the establishment of the amplification system.
[0064] 2. Optimization of real-time fluorescent RPA reaction temperature [00651 The reaction system of real-time fluorescent RPA detection included: buffer A, buffer
B, forward primer FH-F, reverse primer FH-R, probe, and template.
[0066] In this example, the reaction system for real-time fluorescence RPA detection was: 29.5 ul buffer A, 2.5 pL buffer B, 10 uM forward primer FH-F 2 pL, 10 pM reverse primer FH-R 2 ul, 10 uM probe 0.6 ul, template 1 pl (template concentration > 10 fg/ul), made up to 50 ul with deionized water. 10067] Under the conditions of the aforementioned reaction system, MP genomic DNA was added as a template and MP primers and probes designed in Example 1 (Table 1) were added, and the template concentration was 10 pg/uL. The reaction was carried out under constant temperature conditions (five reaction temperatures of 37°C, 38°C, 39°C, 40°C, and 41°C were set), and the results were detected by a real-time thermal cycler, and different dynamic curves were obtained at different temperatures (FIG. 1). The results showed that under the condition of 37-41°C, the RPA amplification efficiency was relatively high and there was no significant difference. In the present invention, 39°C was selected as the constant temperature condition for
RPA amplification. FIG. 1 showed the temperature dynamic curve of the MP RPA primers designed for MP-specific gene sequences.
[0068] Example 3, Establishment of the real-time fluorescent RPA detection method
[0069] 1. Extraction of genomic DNA from the sample to be tested
[0070] The genomic DNA of the samples to be tested was extracted using a DNA extraction kit from Qiagen (QHAamp DNA mint kits; Qiagen, Hilden, Germany) according to the instructions. The concentration and purity of genomic DNA were determined using a UV
S spectrophotometer, and the extracted genomic DNA was aliquoted in small quantities and stored at -20°C for later use.
[0071] 2. Real-time fluorescent RPA detection method
[0072] The genomic DNA extracted in step 1 was used as a template, and the primer FH-F, primer FH-R and probe designed in Example 1 were used to perform real-time fluorescence RPA amplification reaction on the sample to be tested, wherein:
[0073] The real-time fluorescence RPA reaction system was as follows: 29.5 ul buffer A, 2.5 pl buffer B, 10 uM forward primer FH-F 2 ul, 10 uM reverse primer FH-R 2 ul, 19 uM probe 0.6 pL, template 1 pl (template concentration >10fg/ul}, made up to 50 ul with deionized water.
[0074] The reaction conditions of the real-time fluorescence RPA were as follows: the constant temperature was 39 °C, and the fluorescence value of the FAM channel was collected every 308 for a total of 40 cycles; the reaction time was 20 min. [00751 3. Result judgment
[0076] The method for determining whether the sample to be tested contained Mycoplasma pneumoniae or whether it was Mycoplasma pneumoniae according to the amplification product obtained in step 2 was shown as below: [00771 If the sample to be tested showed UNDET or no typical S-shaped amplification curve, the report was negative; if the sample to be tested showed a typical "S-shaped" amplification curve, the CT value of the FAM channel was 37-40, and it needed to be re-tested again; if the test result was still 37-40, it was reported as negative,
[9078] If the sample to be tested showed a typical *S-shaped” amplification curve, the CT value of the FAM channel showed <37, and the report was positive.
[0079] Example 4, the sensitivity of real-time fluorescent RPA detection method
[0080] The serially diluted MP genomic DNA in step 1 of Example 2 was used as a template, and real-time fluorescent RPA detection was performed according to steps 2 and 3 in Example 3.
The results were shown in FIG. 2.
[0081] The real-time fluorescent RPA detection results showed that the senally diluted MP
S genomic DNA was used as the template for RPA amplification reaction. When the amount of
MP genomic DNA template was 100 pg/uL-10 fo/ul., the amplification curve was S-shaped, indicating positive amplification (FIG. 2}. When the amount of genomic template in the reaction system decreased to less than 10 fg, the amplification curve did not rise, indicating a negative result (FIG. 2}. Therefore, the detection range of the real-time fluorescent RPA of the present invention was 100 pg/ul. to 10 fg/ul., indicating that the real-time fluorescent RPA detection method of the present invention had high sensitivity.
[0082] Example 5, the specificity of real-time fluorescent RPA detection method
[9083] In this example, the DNA of common respiratory pathogens and other Mycoplasmas was used as a template to evaluate the specificity of the real-time fluorescent RPA detection method.
[0084] The stram information of the samples to be tested (MP and other pathogenic microorganisms) in this example was shown in Table 2.
[0085] Table 2. Strain information table
Strain Strain Source Strain Number RPA
Results ‘Mycoplasma pneumoniae MI20 1 positive
Mycoplasma genitalium ATCC33530 i negative
Oral Mycoplasma ATCC23714 i negative
Mycoplasma hominis ATCC23114 i negative
Penetrating mycoplasma ATCC55252 i negative
Mycoplasma primate ATCC25960 i negative i3
Streptococcus pneumoniae Isolate (BCH) 1 negative
Staphylococcus aureus Isolate (BCH) i negative
Klebsiella pneumoniae Isolate (BCH) i negative
Pseudomonas aeruginosa Isolate (BCH) 1 negative
Mycobacterium tuberculosis Isolate (BCH) 1 negative
Haemophilus influenzae Isolate (BCH) 1 negative
Acinetobacter baumannii Isolate (BCH) 1 negative
Stenctrophomonas maltophilia Isolate (BCH) 1 negative
Pertussis bacillus Isolate (BCH) 1 negative
Legionella Isolate (BCH) i negative
Respiratory syncytial virus Isolate (BCH) i negative
Adenovirus type 3 Isolate (BCH) i negative
Rhinovirus Isolate (BCH) i negative
HIN influenza virus Isolate (BCH) i negative
[0086] The Mycoplasma isolates in Table 2 were donated by Teacher Zhao Fei from the
Chinese Center for Disease Control and Prevention, and other pathogenic isolates were isolated from the laboratory of Beijing Children's Hospital. The public can obtain this biological material from the applicant, and this biological material was only used for repeating the experiments of the present invention, and cannot be used for other purposes.
[0087] The sample to be tested was detected by real-time fluorescent RPA according to the method of Example 3, and the results were shown in FIG. 3.
[0088] The specificity of the real-time fluorescent RPA detection method was evaluated by using the genomic DNA of common respiratory pathogens and other Mycoplasmas
{Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Haemophilus influenzae, Acinetobacter baumannii,
Stenotrophomonas maltophilia, Pertussis baciilus, Legionella, Respiratory Syncytial Virus,
Adenovirus Type 3, Rhinovirus, HINT Influenza Virus, Mycoplasma genitalium, Oral
S Mycoplasma, Mycoplasma hominis, Penetrating mycoplasma, Mycoplasma Primate) as templates to evaluate the specificity of the real-time fluorescent RPA detection method, and the results showed that only the sample to be tested MP produced a typical amplification curve, and other samples to be tested had no amplification curve, indicating that the real-time fluorescent
RPA detection method of the present invention can accurately identify MP | indicating that the real-time fluorescent RPA detection method of the present invention had good specificity.
[0089] Example 6. Clinical use of real-time fluorescent RPA detection method for MP detection
[0090] The sputum samples of 190 children with community-acquired pneumonia suspected of
MP infection were collected, and the real-time fluorescence RPA detection method and the Real-
IS time PCR detection method were used to conduct MP detection on the aforementioned samples, and the Real-time PCR detection results were used as a standard to evaluate the diagnostic performance of the real-time fluorescent RPA detection method of the present invention for MP.
[0091] Real-time PCR detection methods were as follows: { 1) Extraction of genomic DNA from the sample to be tested 20 The steps were the same as those of step 1 in Example 3. (23 Real-time PCR detection method
The genomic DNA extracted in step (1) was used as a template, and the sample to be tested was amplified by the Real-time PCR detection method, wherein, the Real-time PCR detection adopted a finished kit, which was purchased from Jiangsu Mole Biotechnology Ltd. 25 [0092] The Real-time PCR reaction system was: 6.0 pL buffer, 2.0 uL primer probe, 0.5 pul enzyme, 5.0 pL template, 11.5 pl water, and the total reaction volume was 25 pL. [00931 Real-time PCR reaction conditions were: 50 °C for 2 min, 95 °C for 2 min, 91 °C for s for 40 cycles, and 64 °C for 1 min,
[0094] The negative control was the reaction system without the genome template of
Mycoplasma pneumoniae.
[0095] The positive control was the reaction system containing the genome template of
Mycoplasma pneumoniae P1. 5 [0096] (3) Result judgment
[0097] If the sample to be tested showed UNDECT or no typical S amplification curve, it was a negative sample; when the FAM fluorescence Ct value of the sample to be tested was greater than or equal to 35.33, it was a negative sample.
[9098] If the FAM fluorescence Ct value of the sample to be tested was less than 35.33, and it showed a typical S-shaped amplification curve, it was a positive sample. [00991 The above Real-time PCR detection method and the real-time fluorescent RPA detection method of the present invention {the method of Example 3) were used to detect 190 clinical specimens, and the results were shown in Table 3.
[00100] Table 3. Clinical evaluation of real-time fluorescent RPA and Real-time PCR for MP detection
Diagnostic performance of
RPA Real-time PCR RPA compared to Real-time
Total Kappa PCR
Positive Negative Sensitivity Specificity
Positive 88 4 92 0,958
Negative 0 98 98 100% 96.1%
Total 88 102 190
[00101] The results showed that among the 190 specimens, 92 specimens were positive for real- time fluorescence RPA, 88 were positive for Real-time PCR, and 4 were negative for Real-time
PCR; 98 were negative for both real-time fluorescence RPA and Real-time PCR. With Real-time
PCR as the gold standard, the sensitivity and specificity of real-time fluorescent RPA for detecting MP were 100% and 96.1% respectively.
io
[00102] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experunentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the present invention has been given particular
S embodiments, it should be understood that the present invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the present invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The use of some of the essential features can be made within the scope of the following appended claims.
[00103] In the following paragraphs, clauses are provided:
Clause 1. A reagent or kit for detecting Mycoplasma pneumoniae, characterized in that the reagent or kit comprises a composition for detecting Mycoplasma pneumoniae, the composition comprises a primer pair and a probe, the primer pair consists of a primer FH-F and a primer FH-
ISR, the primer FH-F 18 a single-stranded DNA molecule shown in SEQ ID No.1; and the primer
FH-R is a single-stranded DNA molecule shown in SEQ ID No.2.
Clause 2. The reagent or kit according to clause 1, characterized in that the structure of the probe is nucleotide fragment 1-tetrahydrofuran-nucleotide fragment 2, wherein the nucleotide sequence of nucleotide fragment 1 is SEQ ID No. 3, and the nucleotide sequence of nucleotide fragment 2 is SEQ ID No. 4
Clause 3. The reagent or kit according to clause 2, characterized in that the 3' end of the nucleotide fragment 1 is labeled with a fluorophore, and the 5' end of the nucleotide fragment 2 18 labeled with a quenching group.
Clause 4. The reagent or kit according to clause 3, characterized in that the fluorophore is 6-
FAM, and the quenching group is BHQ1.
Clause 5. A primer pair consisting of a primer FH-F and a primer FH-R, wherein the primer FH-
F is a single-stranded DNA molecule shown in SEQ ID No.1; and wherein the primer FH-R is a single-stranded DNA molecule shown in SEQ ID No.2.
Clause 6. A composition of a probe and the primer pair of clause 5, preferably further comprising any of the features of clauses 2-4.
Clause 7. Use of the primer pair according to clause 5 and/or the composition according to clause 6 in any ong of the following:
A1) use in the detection of Mycoplasma pneumoniae or in the preparation of a product for the detection of Mycoplasma pneumoniae,
AZ) use in identifying or assisting the identification of Mycoplasma pneumoniae or in the preparation of a product for identifying or assisting the identification of Mycoplasma pneumoniae;
A3) use in diagnosing or assisting in the diagnosis of diseases caused by Mycoplasma pneumoniae infection or in the preparation of a product for diagnosing or assisting in the diagnosis of diseases caused by Mycoplasma pneumoniae infection,
Ad) use in screening diseases caused by Mycoplasma pneumoniae infection or in the preparation of a product for screening diseases caused by Mycoplasma pneumoniae infection;
AS} use in the prognosis assessment of diseases caused by Mycoplasma pneumoniae infection or in the preparation of a product for the prognosis assessment of diseases caused by
Mycoplasma pneumoniae infection;
A6) use in therapeutic monitoring of diseases caused by Mycoplasma pneumoniae infection or in the preparation of a product for therapeutic monitoring of diseases caused by
Mycoplasma pneumoniae infection.
Clause 8 A method for detecting Mycoplasma pneumoniae, characterized in that the method comprises using the primer pair according to clause 5 or the composition according to any one according to clauses 6 to carry out a recombinase polymerase amplification reaction on the sample to be tested, and determine whether the sample to be tested contains Mycoplasma pneumoniae or whether the sample to be tested is Mycoplasma pneumoniae according to the amplification product.
Clause 9. The method according to clause 8, characterized in that the temperature of the recombinase polymerase amplification reaction is 37-41°C.
Clause 10. The method according to clause 9, characterized in that the temperature of the recombinase polymerase amplification reaction is 39°C.
Clause 11. The method according to any one according to clauses 8-10, characterized in that the method for determining whether the sample to be tested contains Mycoplasma pneumoniae or whether the sample to be tested is Mycoplasma pneumoniae according to the amplification product is as follows: if the sample to be tested shows UNDET or no typical S-shaped amplification curve, the report is negative, if the sample to be tested shows a typical “S-shaped” amplification curve, the CT value of the FAM channel is 37-40, and it needs to be re-tested again, if the test result is still 37-40, it is reported as negative; if the sample to be tested shows a typical "S-shaped" amplification curve, the CT value of the FAM channel shows <37, and the report is positive.
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| US20160237479A1 (en) * | 2015-02-13 | 2016-08-18 | The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services | Methods and compositions for isothermal amplification and detection of mycoplasma pneumoniae |
| CN109706256A (en) * | 2019-03-01 | 2019-05-03 | 博迪泰（厦门）生物科技有限公司 | A kind of primer, kit and method of inspection detecting mycoplasma pneumoniae MP |
| CN111154916A (en) * | 2020-01-22 | 2020-05-15 | 福建省立医院 | Primer group, detection reagent and kit for respiratory tract pathogen multiple RPA detection |
| CN112592992A (en) * | 2020-12-30 | 2021-04-02 | 济南国益生物科技有限公司 | Primer probe set and kit for combined detection of mycoplasma pneumoniae and chlamydia pneumoniae based on fluorescent RMA method |
| WO2022173266A1 (en) * | 2021-02-15 | 2022-08-18 | 사회복지법인 삼성생명공익재단 | Recombinase-polymerase isothermal amplification device for performing isothermal amplification and simultaneously generating plasmon fluorescence amplification signals |
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2022
- 2022-09-01 CN CN202211063777.3A patent/CN117625814A/en active Pending
- 2022-12-29 NL NL2033870A patent/NL2033870B1/en active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160237479A1 (en) * | 2015-02-13 | 2016-08-18 | The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services | Methods and compositions for isothermal amplification and detection of mycoplasma pneumoniae |
| CN109706256A (en) * | 2019-03-01 | 2019-05-03 | 博迪泰（厦门）生物科技有限公司 | A kind of primer, kit and method of inspection detecting mycoplasma pneumoniae MP |
| CN111154916A (en) * | 2020-01-22 | 2020-05-15 | 福建省立医院 | Primer group, detection reagent and kit for respiratory tract pathogen multiple RPA detection |
| CN112592992A (en) * | 2020-12-30 | 2021-04-02 | 济南国益生物科技有限公司 | Primer probe set and kit for combined detection of mycoplasma pneumoniae and chlamydia pneumoniae based on fluorescent RMA method |
| WO2022173266A1 (en) * | 2021-02-15 | 2022-08-18 | 사회복지법인 삼성생명공익재단 | Recombinase-polymerase isothermal amplification device for performing isothermal amplification and simultaneously generating plasmon fluorescence amplification signals |
Non-Patent Citations (2)
| Title |
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| NIE MING-ZHU ET AL: "Development of a duplex recombinase-aided amplification assay for direct detection of Mycoplasma pneumoniae and Chlamydia trachomatis in clinical samples", JOURNAL OF MICROBIOLOGICAL METHODS, ELSEVIER, AMSTERDAM, NL, vol. 198, 30 May 2022 (2022-05-30), XP087094578, ISSN: 0167-7012, [retrieved on 20220530], DOI: 10.1016/J.MIMET.2022.106504 * |
| ZHAO FLIU ZGU YYANG YXIAO DTAO XMENG FHE LZHANG J: "Detection of Mycoplasma pneumoniae by colorimetric loop-mediated isothermal amplification", ACTA MICROBIOL IMMUNOL HUNG, vol. 60, no. 1, March 2013 (2013-03-01), pages 1 - 9 |
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| CN117625814A (en) | 2024-03-01 |
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