Mycoplasma pneumoniae detection kit and detection method

Abstract

The invention provides a mycoplasma pneumoniae test kit, which comprises a primer probe combination, the primer probe combination comprises a forward primer, a reverse primer and a probe, the forward primer is selected from a group consisting of nucleotide sequences shown in SEQ ID NO: 2-3 and complementary nucleotide sequences thereof, the reverse primer is selected from a group consisting of nucleotide sequences shown in SEQ ID NO: 2-3, and the probe is selected from a group consisting of nucleotide sequences shown in SEQ ID NO: 2-3 and complementary nucleotide sequences thereof. The reverse primer is selected from a group consisting of nucleotide sequences as shown in SEQ ID NO: 6-8 and complementary nucleotide sequences thereof, and the probe is a nucleotide sequence as shown in SEQ ID NO: 9 or a complementary nucleotide sequence thereof. The invention also provides a method for detecting mycoplasma pneumoniae by using the detection kit.

Description

TECHNICAL FIELD

[0001] The present application relates to a Mycoplasma pneumoniae detection kit and a detection method. BACKGROUND

[0002] Mycoplasma pneumoniae is one of the common causes of respiratory infections. Generally, the symptoms of infection are mild, and the symptoms are similar to common cold, such as sore throat, headache, fever, and cough lasting for several weeks to several months. If the infection is in children under the age of five or in adults with low immunity, it can increase symptoms such as nasal congestion, nasal discharge, wheezing, vomiting, and diarrhea. Mild infections can heal on their own, but severe infections can develop into atypical pneumonia, increasing symptoms such as chills, mucous sputum, chest pain, and difficulty breathing. In addition, it can also cause other serious complications, such as severe pneumonia, encephalitis, hemolytic anemia, renal dysfunction, and skin lesions.

[0003] Since Mycoplasma pneumoniae does not have a cell wall, common antibiotics that inhibit cell wall synthesis, such as beta-lactam antibiotics, are ineffective against Mycoplasma pneumoniae, so the preferred treatment is macrolide antibiotics and alternative drugs quinolones or tetracycline antibiotics. To avoid the increase in drug-resistant bacteria caused by antibiotic abuse, it is necessary to test and confirm the pathogenic bacteria before precise medication.

[0004] Current methods for detecting Mycoplasma pneumoniae include bacterial culture, cold agglutinin, serological testing, and nucleic acid detection. Nucleic acid detection has higher sensitivity and specificity and faster results, which is beneficial for physicians to make judgments. Although the nucleic acid detection kits on the market have a certain degree of specificity, the probability of false positives or false negatives is still not low, and most of the nucleic acid detection kits on the market still require about 1 hour or even longer to perform nucleic acid amplification and detection, affecting the accuracy of medication and delaying the treatment opportunity.

[0005] Therefore, it is necessary to provide a nucleic acid detection kit and method different from the past to solve the problems existing in the prior art. SUMMARY

[0006] Therefore, a main object of the present application is to provide a Mycoplasma pneumoniae detection kit, comprising a primer probe combination, which is composed of a forward primer, a reverse primer and a probe, wherein the forward primer is selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 2-3 and the complementary nucleotide sequences thereof, the reverse primer is selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 6-8 and the complementary nucleotide sequences thereof, and the probe is the nucleotide sequence shown in SEQ ID NO: 9 or the complementary nucleotide sequence thereof.

[0007] In some embodiments, the forward primer is the nucleotide sequence shown in SEQ ID NO: 2, and the reverse primer is the nucleotide sequence shown in SEQ ID NO: 8.

[0008] In some embodiments, the 5' end of the probe is connected with a fluorescent reporter group, and the 3' end is connected with a quencher group.

[0009] In some embodiments, the fluorescent reporter group is Cy5.

[0010] In some embodiments, the primer probe combination is in the form of a freeze-dried reagent.

[0011] Another object of the present application is to provide a Mycoplasma pneumoniae detection method, comprising the following steps: S110: providing a biological sample; S120: providing a Mycoplasma pneumoniae detection kit; S130: mixing the biological sample and the Mycoplasma pneumoniae detection kit to obtain a reaction solution; S140: performing a polymerase chain reaction on the reaction solution to amplify the nucleic acid in the biological sample, to obtain an amplification product; S150: detecting a fluorescent signal after each amplification cycle, analyzing the amplification product according to the fluorescent signal to determine whether Mycoplasma pneumoniae exists in the biological sample, wherein when the fluorescent signal is detected, it is determined that Mycoplasma pneumoniae exists in the biological sample; when the fluorescent signal is not detected, it is not determined whether Mycoplasma pneumoniae exists in the biological sample, and the next amplification cycle is performed.

[0012] In some embodiments, the polymerase chain reaction is a real-time quantitative polymerase chain reaction (qPCR).

[0013] In some embodiments, the polymerase chain reaction comprises 40-50 amplification cycles.

[0014] In some embodiments, each of the amplification cycles comprises: a denaturation reaction at 93-97°C for 4-6 seconds; and an annealing and extension reaction at 58-62°C for 8-12 seconds.

[0015] In some embodiments, an enzyme activation reaction at 93-97°C for 25-35 seconds is performed before the amplification cycles.

[0016] Compared to the prior art, the present application has the following advantages: (1) high specificity: the sequence coverage rate of the primers and probes is maintained at more than 99%, and no gene sequence fragments of other species other than Mycoplasma pneumoniae are recognized, with high specificity; (2) high sensitivity: the detection limit of the primer probe combination is 5 copy / rxn of nucleic acid sample; and when there is human genomic nucleic acid interference, the primer probe combination can also detect 100 copy / rxn of nucleic acid sample; (3) fast reaction time: compared with other test kits in the industry, the reaction time of the test kit of the present application is less than 1 hour, and the test result can be obtained quickly; and (4) cost reduction: the primer probe combination is freeze-dried, which does not affect the reaction efficiency and can reduce the cold chain cost during transportation. BRIEF DESCRIPTION OF DRAWINGS

[0017] For a more complete understanding of the embodiments and their advantages, reference is now made to the following description taken in conjunction with the accompanying drawings in which: Figure 1 is a flow chart of the detection method of Mycoplasma pneumoniae of the present application; Figure 2 shows a partial sequence of the P1 gene of Mycoplasma pneumoniae and the design position of the primers and probes; Figure 3A and 3B are respectively the amplification curve graph and the standard curve graph obtained after the qPCR reaction of the Mycoplasma pneumoniae test kit of the present application; and Figure 4 is the gel electrophoresis graph of the product after the qPCR reaction of the Mycoplasma pneumoniae test kit of the present application. DETAILED DESCRIPTION

[0018] Particular embodiments according to the present application will be described below; however, the present application can be practiced in a variety of different forms without departing from the spirit of the present application, and the scope of protection of the present application should not be interpreted as being limited to what is stated in the specification. In addition, unless otherwise stated in the text, "a", "the", and similar terms used in the specification (especially in the claims below) should be understood to include both singular and plural forms.

[0019] Although the following detailed description utilizes a series of operations or steps to illustrate the methods disclosed herein, the order of the operations or steps shown is not to be construed as a limitation of the present disclosure. For example, certain operations or steps can be performed in different order and / or concurrently with other steps. In addition, not all of the operations, steps, and / or features can be necessary to practice an embodiment of the present disclosure. Furthermore, each of the operations or steps described herein can include sub-steps or actions.

[0020] An embodiment of the present disclosure provides a Mycoplasma pneumoniae test kit, comprising a primer probe combination, which is composed of a forward primer, a reverse primer, and a probe, wherein the forward primer is selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 2-3 and the complementary nucleotide sequences thereof, the reverse primer is selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 6-8 and the complementary nucleotide sequences thereof, and the probe is the nucleotide sequence shown in SEQ ID NO: 9 or the complementary nucleotide sequence thereof.

[0021] In a specific embodiment, the present disclosure uses SEQ ID NO: 2 as the forward primer, and SEQ ID NO: 8 as the reverse primer, in combination with the probe sequence of SEQ ID NO: 9 as the primer probe combination in the Mycoplasma pneumoniae test kit.

[0022] In a specific embodiment, the 5' end of the probe is attached to a fluorescent reporter group, and the 3' end is attached to a quencher group. The types of the fluorescent reporter group and the quencher group are not limited, as long as they can stably emit a fluorescent signal and do not affect the function of the probe. For example, the fluorescent reporter group can be Cy5.

[0023] Another embodiment of the present disclosure provides a detection method for Mycoplasma pneumoniae, as shown in Figure 1 The detection method comprises the following steps: S110: providing a biological sample; S120: providing the aforementioned Mycoplasma pneumoniae test kit; S130: mixing the biological sample and the Mycoplasma pneumoniae test kit to obtain a reaction solution; S140: performing a polymerase chain reaction on the reaction solution to amplify the nucleic acid in the biological sample to obtain an amplification product; and S150: detecting the fluorescence signal after each amplification cycle, analyzing the amplification product according to the fluorescence signal to determine whether the Mycoplasma pneumoniae exists in the biological sample, wherein when the fluorescence signal is detected, it is determined that the Mycoplasma pneumoniae exists in the biological sample; when the fluorescence signal is not detected, it is not determined whether the Mycoplasma pneumoniae exists in the biological sample, and the next amplification cycle is performed.

[0024] The Mycoplasma pneumoniae test kit of the present application can be used for various polymerase chain reaction-based technologies. In a specific embodiment, the polymerase chain reaction can be a real-time quantitative polymerase chain reaction. For example, the polymerase chain reaction can include 45 amplification cycles, each of which can include a denaturation reaction at 95°C for 5 seconds, and an annealing and extension reaction at 60°C for 10 seconds. Preferably, an enzyme activation reaction can be performed before the amplification cycle, which can be at 95°C for 30 seconds.

[0025] To further demonstrate that various embodiments of the present application can be used to detect the presence of Mycoplasma pneumoniae and have various advantages described above, the following tests were performed.

[0026] Preparation Example 1: Design of primers and probes

[0027] According to the information provided by the GenBank database about the Mycoplasma pneumoniae P1 gene (accession number: LC753471.1), Figure 2 a partial sequence of the Mycoplasma pneumoniae P1 gene is shown, and the design position of the primers and probes is shown.

[0028] In this embodiment, the forward primer is the nucleotide sequence shown in SEQ ID NO: 1-4, which is complementary to the sequence after the 5421st base of the Mycoplasma pneumoniae P1 gene (as shown by the left arrow); Figure 2 the reverse primer is the nucleotide sequence shown in SEQ ID NO: 5-8, which is complementary to the sequence before the 5553rd base of the Mycoplasma pneumoniae P1 gene (as shown by the right arrow). Figure 2

[0029] In this embodiment, the probe is the nucleotide sequence shown in SEQ ID NO: 9, which is complementary to the sequence between the 5471st base and the 5494th base of the Mycoplasma pneumoniae P1 gene (as shown by the middle arrow), and has Cy5 at its 5' end. Figure 2

[0030] Preparation Example 2: Preparation of Mycoplasma pneumoniae P1 gene plasmid

[0031] According to Figure 2 ​​The LC753471.1 sequence shown was cloned into a vector to obtain a standard plasmid with the Mycoplasma pneumoniae P1 gene (hereinafter referred to as "P1 template"), and a plasmid containing only the vector was used as a control group (hereinafter referred to as "control group") for subsequent experiments. In accordance with the present application, any suitable vector can be used to construct a standard plasmid with the Mycoplasma pneumoniae P1 gene, as long as it is replicable.

[0032] Preparation Example 3: Screening of primers and probes

[0033] A. Sequence retention of primers and probes

[0034] The nucleic acid sequences of the forward primers, reverse primers and probes designed according to the Mycoplasma pneumoniae P1 gene (i.e., SEQ ID NO: 1-9) were analyzed by Nucleotide Blast in NCBI, and the primer sequences were analyzed by Primer Blast. As shown in Table 1, the analysis results confirmed that the sequence retention of SEQ ID NO: 1-9 can reach 99.1 or more, with good specificity, excluding the possibility of other species nucleic acid sequence fragments being identified.

[0035] Table 1

[0036] B. Screening of primer and probe sequences In order to screen the primer and probe combination with the best detection performance (Set 1 to Set 15 shown in Table 2) from the above-mentioned forward primers, reverse primers and probes (shown in Table 1), 10 6 The P1 template with copy number (cp) was diluted 10-fold to prepare the following copy numbers: 10 4 cp, 10 2 cp, 10 1cp; the forward primer, the reverse primer and the probe in Set 1 to Set 15 and the qPCR reaction reagent were added into the P1 template of each of the aforementioned various copy numbers to generate a reaction solution; the reaction solution was placed into a qPCR machine, and first maintained at 95°C for 30 seconds for enzyme activation stage, and then performed 45 cycles of PCR stage, each cycle using 95°C denaturation for 5 seconds and 60°C annealing and extension for 10 seconds, and fluorescence detection was performed at the end of each cycle. The overall reaction time of PCR was 48 minutes. After the reaction was completed, the threshold cycle (quantification cycle, Cq) and amplification efficiency of Set 1 to Set 15 were compared. As can be seen from Table 2, the groups with better amplification efficiency and earlier Cq value (i.e. faster reaction time) when the copy number was low were Set 1, Set 3, Set 5, Set 6, Set 7, Set 9 and Set 15. The aforementioned groups were then subjected to a second round of screening (as shown in Table 3). Human genome DNA (gDNA) was mixed with the P1 template, and the qPCR reaction was performed with the primer probe combination of each group in Table 3 as described in Preparation Example 2. The P1 template without the addition of human gDNA was used as the gDNA-free control group. Each group was subjected to two repeats, and the situation under the interference of human gDNA was observed. As can be seen from Table 3, Set 5, Set 6, Set 7 and Set 9 were not interfered by human gDNA. Considering the time of Cq value generation, the range of ΔCq, amplification efficiency and whether the control group had abnormal signals, Set 7 was selected for subsequent testing because it had the earliest Cq value generation, the ΔCq range was within ±1, the amplification efficiency was the best and the control group had no abnormal signals. The primer probe combination of Set 7 (SEQ ID No: 2 + SEQ ID No: 8 + SEQ ID No: 9) was selected for subsequent testing.

[0037] Table 2 #: the signal had abnormal rising, which might affect the detection specificity and cause false positive; ND: Non-Detect.

[0038] Table 3

[0039] Example 1: Sensitivity analysis of the Mycoplasma pneumoniae detection kit of the present application

[0040] In this example, the sensitivity of the Mycoplasma pneumoniae detection kit containing the specific primer probe combination (SEQ ID No: 2 + SEQ ID No: 8 + SEQ ID No: 9) of the present application was tested using P1 templates with different copy numbers. First, 10 6P1 template of 20 cp, 100 cp, 10,000 cp, respectively, and then diluted by 10 times to prepare the following copy numbers: 20 cp, 100 cp, 10,000 cp 5 P1 template of 20 cp, 100 cp, 10,000 cp, respectively, and then diluted by 10 times to prepare the following copy numbers: 20 cp, 100 cp, 10,000 cp 4 P1 template of 20 cp, 100 cp, 10,000 cp, respectively, and then diluted by 10 times to prepare the following copy numbers: 20 cp, 100 cp, 10,000 cp 3 P1 template of 20 cp, 100 cp, 10,000 cp, respectively, and then diluted by 10 times to prepare the following copy numbers: 20 cp, 100 cp, 10,000 cp 2 P1 template of 20 cp, 100 cp, 10,000 cp, respectively, and then diluted by 10 times to prepare the following copy numbers: 20 cp, 100 cp, 10,000 cp 1 P1 template of 20 cp, 100 cp, 10,000 cp, respectively, and then diluted by 10 times to prepare the following copy numbers: 20 cp, 100 cp, 10,000 cp

[0041] Referring to Figure 2 A 3A and 2B 3B , which are amplification curve and standard curve obtained after qPCR reaction. As shown in Figure 2 A 3A , there is no amplification product (i.e., the horizontal line in the lower part of Figure 2 A 3A , so there is no fluorescence reaction. On the contrary, 10 1 cp to 10 6 cp show positive fluorescence reaction results.

[0042] With continued reference to Figure 2 B 3B , the amplified results of Figure 2 A 3A are made into a standard curve. The horizontal axis is the logarithm of the copy number, which is the logarithm of the starting quantity (Log starting quantity), and the vertical axis is the Cq value. Generally, the amplification efficiency is between 90% and 110%. As shown in Figure 2 B 3B , according to the results of 10 1 cp to 10 6 cp, a straight line with a slope of 3.179 can be drawn, with a correlation coefficient of 0.996 and an amplification efficiency of 106.3%. Therefore, the sensitivity of the Mycoplasma pneumoniae test kit comprising the specific primer probe combination (SEQ ID No: 2 + SEQ ID No: 8 + SEQ ID No: 9) of the present application meets the range of amplification efficiency.

[0043] Example 2: Specificity analysis of the Mycoplasma pneumoniae test kit of the present application

[0044] This example tests the specificity of the Mycoplasma pneumoniae test kit comprising the specific primer probe combination (SEQ ID No: 2 + SEQ ID No: 8 + SEQ ID No: 9) of the present application by qPCR reaction as described in Preparation Example 2. P1 templates with the following copy numbers were prepared: 20 cp, 100 cp, 10,000 cp, and qPCR reaction was performed, and the amplification products obtained were analyzed by gel electrophoresis, with the results shown in Figure 3.

[0045] As shown in FIG. 3, the results of 0 cp did not exist any amplification product, the amplification product size of 20 cp, 100 cp and 10000 cp was about 109 bp, and no non-specific product existed. Therefore, the Mycoplasma pneumoniae test kit of the present application comprising the specific primer probe combination (SEQ ID No: 2 + SEQ ID No: 8 + SEQ ID No: 9) has high specificity, and no non-specific amplification occurs.

[0046] Example 3: Interference test of the Mycoplasma pneumoniae test kit of the present application

[0047] In order to detect whether human gDNA would interfere with the detection of Mycoplasma pneumoniae and affect the test results, 200 ng of human gDNA was added to the biological sample containing Mycoplasma pneumoniae, and the DELBio G1-Automatic Nucleic Acid Testing was sequentially performed for Mycoplasma pneumoniae gene extraction and purification, Mycoplasma pneumoniae gene qPCR amplification, and fluorescence detection and result analysis, and two repeated tests were performed. As shown in Table 4, the results showed that compared with the control group, the 1000 copy / rxn biological sample containing Mycoplasma pneumoniae, the 100 copy / rxn biological sample containing Mycoplasma pneumoniae and the 20 copy / rxn biological sample containing Mycoplasma pneumoniae could be detected by the Mycoplasma pneumoniae test kit of the present application, and the Cq values of the 100 copy / rxn biological sample containing Mycoplasma pneumoniae and the 100 copy / rxn biological sample containing Mycoplasma pneumoniae added with 200 ng of human gDNA were not different, showing that the Mycoplasma pneumoniae test kit of the present application comprising the specific primer probe combination (SEQ ID No: 2 + SEQ ID No: 8 + SEQ ID No: 9) could detect the biological sample containing Mycoplasma pneumoniae under different copy numbers and 200 ng gDNA interference, and the detection sensitivity under 200 ng gDNA interference could also detect 100 copy / rxn.

[0048] Table 4

[0049] Example 4: Freeze-dried reagent performance test of the Mycoplasma pneumoniae test kit of the present application

[0050] To determine whether the reagents of the Mycoplasma pneumoniae test kit of the present application containing the specific primer probe combination (SEQ ID No: 2 + SEQ ID No: 8 + SEQ ID No: 9) would be affected after freeze-drying, 100 copies and 20 copies of P1 template were prepared, respectively, and the Mycoplasma pneumoniae test kit in liquid reagent form and the Mycoplasma pneumoniae test kit after reconstitution of freeze-dried reagents were subjected to qPCR reaction as described in Preparation Example 2, and the test was repeated twice, the Cq value and the range of ΔCq were compared, and the results are shown in Table 5.

[0051] As can be seen from Table 5, whether the 100 copies and 20 copies of P1 template were detected by liquid reagents or freeze-dried reagents, there was no difference in Cq value, and the range of ΔCq was within ±1, indicating that the performance of the Mycoplasma pneumoniae test kit of the present application after freeze-drying was not affected.

[0052] Table 5

[0053] Example 5: Limit of Detection (LoD) of the Mycoplasma pneumoniae test kit of the present application

[0054] To determine the limit of detection of the Mycoplasma pneumoniae test kit of the present application containing the specific primer probe combination (SEQ ID No: 2 + SEQ ID No: 8 + SEQ ID No: 9), 20 copy / rxn, 10 copy / rxn and 5 copy / rxn of P1 template were prepared, respectively, and 10 5 copy / rxn of human gene was added as an internal control group, and qPCR reaction was performed using the Mycoplasma pneumoniae test kit of the present application as described in Preparation Example 2, 20 copy / rxn P1 template was subjected to 12 repeated tests, 10 copy / rxn and 5 copy / rxn P1 template was subjected to 6 repeated tests, and finally fluorescence detection (the fluorescence probe of the internal control group was connected with HEX fluorescence group) and result analysis were performed, as shown in Table 6.

[0055] As can be seen from Table 6, 20 copy / rxn P1 template with 10 5 copy / rxn of human gene was subjected to 12 repeated tests, and 10 5The 6-repeated test of the P1 template of 10 copy / rxn and 5 copy / rxn of the human gene can be detected by the Mycoplasma pneumoniae test kit of the present application, which shows that the LoD of the Mycoplasma pneumoniae test kit of the present application containing the specific primer probe combination (SEQ ID No: 2 + SEQ ID No: 8 + SEQ ID No: 9) can be as low as 5 copy / rxn under the condition of the existence of high concentration of human gene. copy / rxn.

[0056] Table 6

[0057] In summary, the advantages of the present application are: (1) High specificity: the sequence coverage rate of the maintained primer and probe is more than 99%, and the gene sequence fragments of other species other than Mycoplasma pneumoniae are not recognized, which has high specificity; (2) High sensitivity: the detection limit of the primer probe combination is 5 copy / rxn of nucleic acid sample; and when there is human genome nucleic acid interference, the primer probe combination can also detect 100 copy / rxn of nucleic acid sample; (3) Fast reaction time: compared with other test kits in the industry, the reaction time of the test kit of the present application is less than 1 hour, and the test result can be obtained quickly; and (4) Reduce cost: the reaction efficiency of the primer probe combination is not affected after freeze-drying treatment, and the cold chain cost in the transportation process can be reduced.

[0058] Although the present application has been disclosed with preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application shall be subject to the appended claims.

Claims

1. A Mycoplasma pneumoniae detection kit comprising a primer probe combination, the primer probe combination consisting of a forward primer, a reverse primer and a probe, wherein the forward primer is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 2-3 and the complementary nucleotide sequences thereof, the reverse primer is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 6-8 and the complementary nucleotide sequences thereof, and the probe is the nucleotide sequence set forth in SEQ ID NO: 9 or the complementary nucleotide sequence thereof. 2.The Mycoplasma pneumoniae detection kit of claim 1, wherein the forward primer is the nucleotide sequence set forth in SEQ ID NO: 2, and the reverse primer is the nucleotide sequence set forth in SEQ ID NO:

8. 3.The Mycoplasma pneumoniae detection kit of claim 1, wherein a fluorescent reporter group is attached to the 5' end of the probe, and a quencher group is attached to the 3' end of the probe. 4.The Mycoplasma pneumoniae detection kit of claim 3, wherein the fluorescent reporter group is Cy5. 5.The Mycoplasma pneumoniae detection kit of claim 1, wherein the primer probe combination is in the form of a freeze-dried reagent. 6.A method for detecting Mycoplasma pneumoniae, comprising the steps of: S110: providing a biological sample; S120: providing the Mycoplasma pneumoniae detection kit according to any one of claims 1 to 5; S130: mixing the biological sample and the Mycoplasma pneumoniae detection kit to obtain a reaction solution; S140: performing a polymerase chain reaction on the nucleic acid in the biological sample to amplify the nucleic acid to obtain an amplification product; and S150: detecting a fluorescent signal after each amplification cycle, analyzing the amplification product according to the fluorescent signal to determine whether Mycoplasma pneumoniae is present in the biological sample, wherein when the fluorescent signal is detected, it is determined that Mycoplasma pneumoniae is present in the biological sample; when the fluorescent signal is not detected, it is not determined whether Mycoplasma pneumoniae is present in the biological sample, and the next amplification cycle is performed. 7.The method for detecting Mycoplasma pneumoniae of claim 6, wherein the polymerase chain reaction is a real-time quantitative polymerase chain reaction. 8.The method for detecting Mycoplasma pneumoniae of claim 6, wherein the polymerase chain reaction comprises 40 to 50 amplification cycles. 9.The method for detecting Mycoplasma pneumoniae of claim 8, wherein each amplification cycle comprises: a denaturation reaction at 93-97℃ for 4-6 seconds; and an annealing and extension reaction at 58-62℃ for 8-12 seconds. 10.The method for detecting Mycoplasma pneumoniae of claim 8, wherein an enzyme activation reaction is performed before the amplification cycle: at 93-97℃ for 25-35 seconds. ​

Images