Primer composition, kit for efficiently detecting common pathogenic bacteria of human and application thereof

Abstract

The application provides a primer composition, a kit and application thereof for efficiently detecting common pathogenic bacteria of human, relates to the technical field of microorganism gene detection, and aims at solving the defects of low detection efficiency, poor specificity, low sensitivity of traditional PCR technology, and non-specific amplification of traditional LAMP detection technology. The application designs primers according to known sequences of six common pathogenic bacteria of human, i.e., Candida albicans, Chlamydia trachomatis, Escherichia coli, group B streptococcus, Mycoplasma hominis and Streptococcus pneumoniae, and provides a kit with a micro-fluidic chip as a carrier and application thereof, which are used for detecting common pathogenic bacteria of human, have high specificity and high sensitivity, reduce detection cost and detection time, and can realize on-site rapid detection of common pathogenic bacteria of human.

Description

Technical Field

[0001] This invention relates to the field of microbial gene detection technology, and more specifically, to a primer composition, kit, and application for the efficient detection of common human pathogens. Background Technology

[0002] Bacteria that can cause human diseases are collectively called pathogenic bacteria. Examples include Candida albicans (CaR), Chlamydia trachomatis (CT), Escherichia coli (E. coli), Group B Streptococcus agalactiae (GBS), Mycoplasma hominis (MH), and Streptococcus pneumoniae. Pneumoniae (SP) are common pathogens. Among them, Candida albicans can cause fungal infections such as oral candidiasis and candidal vaginitis; Chlamydia trachomatis can cause diseases such as trachoma, inclusion body capsulitis, urogenital tract infections, and lymphogranuloma venereum; Escherichia coli is closely related to diarrhea and food poisoning; Group B Streptococcus is one of the important pathogens in postpartum and neonatal infections; Mycoplasma hominis can cause urinary tract infections and genital inflammation; Streptococcus pneumoniae is an opportunistic pathogen that can cause lobar pneumonia, otitis media, bronchitis, meningitis, and sepsis, and is common in infants under 2 years old and the elderly over 65 years old.

[0003] Currently, gene detection has become one of the main methods for detecting pathogens. Among them, gene detection is mainly based on polymerase chain reaction (PCR) detection. PCR technology was proposed by Kary B. Mullis in 1983 and invented in 1985. At present, PCR technology is relatively mature. However, PCR reaction needs to be cycled in two different temperature zones, which requires high-end instruments and is relatively expensive. At the same time, PCR technology only has one pair of amplification primers, which is easily affected by environmental interference and has relatively low specificity. Moreover, PCR technology is difficult to complete large-scale detection in a short time, the result time is long, the operation requires high professional skills, there are many micro-dosing steps, and it is difficult to control on-site detection and rapid detection.

[0004] Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification technique proposed by Notomi et al. in 2000, has been widely applied in pathogen detection and infectious disease diagnosis, showing promising application prospects. Its advantages include high detection sensitivity and accuracy, and the fact that LAMP does not require additional heating and cooling procedures, making it a promising candidate for large-scale on-site and rapid detection. However, due to its isothermal nature, it lacks a hot-start enzyme similar to PCR, which can lead to non-specific amplification during the equipment heating phase, thus affecting the detection results.

[0005] In conclusion, developing an efficient method for detecting pathogenic bacteria can generate significant social and economic benefits. Summary of the Invention

[0006] To address the above problems, this invention provides a primer composition, kit, and application for the efficient detection of common human pathogens.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides primer compositions for detecting six common human pathogens (Candida albicans, Chlamydia trachomatis, Escherichia coli, Group B Streptococcus, Mycoplasma hominis, and Streptococcus pneumoniae), including:

[0009] The upstream external primer (CaR-F3-6), downstream external primer (CaR-B3-6), upstream internal primer (CaR-FIP-6), downstream internal primer (CaR-BIP-6), upstream loop primer (CaR-LF-6), and downstream loop primer (CaR-LB-6) for Candida albicans.

[0010] The upstream external primer (CT-F3-1), downstream external primer (CT-B3-1), upstream internal primer (CT-FIP-1), downstream internal primer (CT-BIP-1), and upstream loop primer (CT-LF-1) for Chlamydia trachomatis.

[0011] The upstream external primer (Eco-F3-1), downstream external primer (Eco-B3-1), upstream internal primer (Eco-FIP-1), downstream internal primer (Eco-BIP-1), upstream loop primer (Eco-LF-1), and downstream loop primer (Eco-LB-1) for Escherichia coli.

[0012] The upstream external primer (GBS-F3-2), downstream external primer (GBS-B3-2), upstream internal primer (GBS-FIP-2), downstream internal primer (GBS-BIP-2), and downstream loop primer (GBS-LB-2) for Group B Streptococcus.

[0013] upstream external primer (MH-F3-3), downstream external primer (MH-B3-3), upstream internal primer (MH-FIP-3), downstream internal primer (MH-BIP-3), upstream loop primer (MH-LF-3);

[0014] The upstream external primer (SP-F3-7), downstream external primer (SP-B3-7), upstream internal primer (SP-FIP-7), downstream internal primer (SP-BIP-7), upstream loop primer (SP-LF-7), and downstream loop primer (SP-LB-7) for Streptococcus pneumoniae.

[0015] The gene sequences of the six primer combinations are shown in SEQ ID NO: 1 to SEQ ID NO: 33.

[0016] Unlike the DNA polymerase used in traditional PCR, LAMP technology utilizes a BstDNA polymerase with high chain displacement activity. Each LAMP primer set includes two outer primers: F3 and B3, two inner primers: FIP and BIP, and two loop-guided primers: LOOP F and LOOP B, where LOOP F and LOOP B are non-essential primers.

[0017] Furthermore, the six primer compositions first search for target sequences through NCBI GenBank, and then LAMP primers are designed and screened for the target sequences.

[0018] Furthermore, the target genes of the six primer sets are as follows: the nucleic acid sequence of Candida albicans is U46158, and the target gene is the RSR1 gene; the nucleic acid sequence of Chlamydia trachomatis is HE603228, and the target gene refers to a partial sequence of the cryptic plasmid gene; the nucleic acid sequence of Escherichia coli is CP054942, and the target gene is the traA gene; the nucleic acid sequence of Group B Streptococcus is CP019814, and the target gene is the cfb gene; the nucleic acid sequence of Mycoplasma hominis is CP055150, and the target gene refers to a partial sequence of the alaS gene; and the nucleic acid sequence of Streptococcus pneumoniae is MK606437, and the target gene is the cps gene.

[0019] This invention also provides a kit for the efficient detection of Candida albicans, Chlamydia trachomatis, Escherichia coli, Group B Streptococcus, Mycoplasma hominis, and Streptococcus pneumoniae.

[0020] Furthermore, this kit includes a microfluidic chip, a reaction solution, and six sets of primers as described above.

[0021] Biochips are a novel detection method that uses methods such as photoconductive in-situ synthesis or micro-spotting to orderly immobilize a large number of biological macromolecules, such as nucleic acid fragments, peptide molecules, carbohydrates, and even tissue sections and cells, on the surface of a support such as a glass slide, silicon wafer, or filter membrane, forming a highly dense two-dimensional molecular arrangement. These molecules then hybridize with target molecules in a labeled biological sample. Specific instruments are used to rapidly, in parallel, and efficiently monitor and analyze the intensity of the hybridization signal, thereby determining the quantity of target molecules in the sample. This method is characterized by high throughput, miniaturization, automation, low cost, and low pollution. Microfluidic chips, also known as lab-on-a-chip, are a new type of biochip. Microfluidic chips can detect PCR products in the range of 15-7500 bp with a resolution of up to 20 bp. Sample miniaturization further reduces diffusion, resulting in excellent separation. Each well can simultaneously analyze multiple different PCR products.

[0022] Furthermore, the microfluidic chip of this invention is an 8-sample chip, meaning one sample well corresponds to four detection wells. One microfluidic chip has four LAMP reaction regions, each containing eight detection wells and two sample wells. The specific structure is as follows: Figure 1 As shown.

[0023] Furthermore, the embedding material for the detection holes of the microfluidic chip is:

[0024] Primers for amplifying the Candida albicans sequence were embedded in the detection wells. The primer composition was as follows: 0.5 μL of 90 μM CaR-F3-6, 0.5 μL of 90 μM CaR-B3-6, 2 μL of 180 μM CaR-FIP-6, 2 μL of 180 μM CaR-BIP-6, 1 μL of 180 μM CaR-LF-6, and 1 μL of 180 μM CaR-LB-6.

[0025] Primers for amplifying the Chlamydia trachomatis sequence were embedded in the detection wells. The primer composition was as follows: 0.5 μL of 90 μM CT-F3-1, 0.5 μL of 90 μM CT-B3-1, 2 μL of 180 μM CT-FIP-1, 2 μL of 180 μM CT-BIP-1, and 1 μL of 180 μM CT-LF-1.

[0026] Primers for amplifying Escherichia coli sequences were embedded in the detection wells. The primer composition was as follows: 0.5 μL of 90 μM Eco-F3-1, 0.5 μL of 90 μM Eco-B3-1, 2 μL of 180 μM Eco-FIP-1, 2 μL of 180 μM Eco-BIP-1, 1 μL of 180 μM Eco-LF-1, and 1 μL of 180 μM Eco-LB-1.

[0027] Primers for amplifying the group B streptococcus sequence were embedded in the detection wells. The primer composition was as follows: 0.5 μL of 90 μM GBS-F3-2, 0.5 μL of 90 μM GBS-B3-2, 2 μL of 180 μM GBS-FIP-2, 2 μL of 180 μM GBS-BIP-2, and 1 μL of 180 μM GBS-LB-2.

[0028] Primers for amplifying the human mycoplasma sequence were embedded in the detection wells of Chlamydia hominis. The primer composition was as follows: 0.5 μL of 90 μM MH-F3-3, 0.5 μL of 90 μM MH-B3-3, 2 μL of 180 μM MH-FIP-3, 2 μL of 180 μM MH-BIP-3, and 1 μL of 180 μM MH-LF-3.

[0029] Primers for amplifying the Streptococcus pneumoniae sequence were embedded in the detection wells. The primer composition was as follows: 0.5 μL of 90 μM SP-F3-7, 0.5 μL of 90 μM SP-B3-7, 2 μL of 180 μM SP-FIP-7, 2 μL of 180 μM SP-BIP-7, 1 μL of 180 μM SP-LF-7, and 1 μL of 180 μM SP-LB-7.

[0030] The blank test well serves as a blank control.

[0031] Internal reference primers and internal reference plasmids were embedded in the internal reference detection wells.

[0032] Furthermore, after the primer kits were embedded in the microfluidic chip, they were freeze-dried and stored.

[0033] Furthermore, the composition of the reaction solution required for each of the 8 test wells in this kit is as follows:

[0034]

[0035] A total of 18 μL was collected and stored separately in the reaction solution container.

[0036] Gold nanoparticles possess high electron density and dielectric properties, allowing them to bind to various biomolecules without affecting their biological activity. They can also specifically enhance fluorescence signals. Addressing the drawback of traditional TAMP technology, which is prone to non-specific amplification during the heating phase, this invention adds gold nanoparticles to the reaction solution to adsorb ssDNA and proteases, effectively suppressing the non-specific reactions in LAMP technology during the heating phase.

[0037] This invention also provides an application of the kit for the efficient detection of common human pathogens, as described above. The specific application method is as follows: Take 38.4 μL of sample, mix it with 18 μL of reaction solution, and add it to two sample wells. Cover the sample wells with sealing film and then perform detection. During detection, the reaction temperature is maintained at 63.5℃, and the reaction time is 30 min. After the reaction, the sample is centrifuged at 1600 rpm for 10 seconds, and at 4600 rpm for 30 seconds. The detection results are determined by interpreting the S-curve of the detector.

[0038] The beneficial effects of this invention are as follows:

[0039] Using LAMP technology instead of traditional PCR technology can detect pathogens more accurately and sensitively. Adding gold nanoparticles to the reaction system adsorbs ssDNA and proteases, inhibiting non-specific reactions during the heating process and achieving hot-start detection. Combining the improved LAMP technology with microfluidic chip technology allows for rapid and accurate detection results, as well as simultaneous detection of multiple different indicators on the same sample. Furthermore, the reaction reagents are pre-embedded in the microfluidic chip, enabling the detection of six indicators simultaneously. Users only need to add the sample; operation is simple. The instrument is equipped with a lithium battery for convenient on-site rapid testing. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the aperture positions of a microfluidic chip;

[0041] Figure 2 This is a diagram showing the amplification results of Example 4;

[0042] Figure 3 This is a diagram showing the amplification results of Example 5;

[0043] Figure 4 This is a diagram showing the amplification results of Example 6.

[0044] Figure labeling: 11-First sample loading well; 12-Second sample loading well; 13-Candida albicans detection well; 14-Chlamydia trachomatis detection well; 15-Escherichia coli detection well; 16-Group B streptococcus detection well; 17-Mycoplasma hominis detection well; 18-Streptococcus pneumoniae detection well; 19-Blank detection well; 110-Internal control detection well. Detailed Implementation

[0045] To make the above-mentioned objectives, features, and advantages of the present invention more apparent and understandable, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Example 1

[0047] This example demonstrates the preparation of LAMP primers.

[0048] First, target sequences were identified using NCBI GenBank. These were: Candida albicans (U46158, RSR1 gene); Chlamydia trachomatis (HE603228, partial sequence of cryptic plasmid gene); Escherichia coli (CP054942, traA gene); Group B Streptococcus (CP019814, cfb gene); Mycoplasma hominis (CP055150, partial sequence of alaS gene); and Streptococcus pneumoniae (MK606437, cps gene).

[0049] LAMP primers were designed using computer software for six target sequences, and primers were synthesized. The synthesized primers were then screened to obtain a set of LAMP primers specifically for microfluidic chips.

[0050] The primer set consists of an upstream external primer, a downstream external primer, an upstream internal primer, and a downstream internal primer. The primer sets for Candida albicans, Escherichia coli, and Streptococcus pneumoniae also include an upstream circular primer and a downstream circular primer. The primer sets for Chlamydia trachomatis and Mycoplasma hominis include an upstream circular primer, and the primer set for Group B Streptococcus includes a downstream circular primer. The sequences of each primer are shown in SEQ ID NO: 1 to SEQ ID NO: 33.

[0051] Example 2

[0052] This embodiment describes the development of a highly efficient reagent kit for detecting human pathogens.

[0053] This kit includes the primer set constructed in Example 1, as well as a microfluidic chip and reaction solution.

[0054] The pore structure of microfluidic chips is as follows Figure 1As shown, a microfluidic chip includes four reaction regions, capable of simultaneously detecting six indicators in four groups of samples. One reaction region consists of two sample loading wells and eight detection wells. The sample loading wells are divided into a first sample loading well 11 and a second sample loading well 12. Primers are embedded in the detection wells. The primer set for Candida albicans is embedded in the Candida albicans detection well 13; the primer set for Chlamydia trachomatis is embedded in the Chlamydia trachomatis detection well 14; the primer set for Escherichia coli is embedded in the Escherichia coli detection well 15; the primer set for Group B Streptococcus is embedded in the Group B Streptococcus detection well 16; the primer set for Mycoplasma hominis is embedded in the Mycoplasma hominis detection well 17; the primer set for Streptococcus pneumoniae is embedded in the Streptococcus pneumoniae detection well 18; the blank control is the blank detection well 19; and the internal control is embedded in the internal control detection well 110.

[0055] The primer sets were embedded in the following quantities: 0.5 μL of 90 μM upstream external primer, 0.5 μL of 90 μM downstream external primer, 2 μL of 180 μM upstream internal primer, 2 μL of 180 μM downstream internal primer, 1 μL of 180 μM upstream loop primer, and 1 μL of 180 μM downstream loop primer. After embedding the primer sets into the detection wells, they were lyophilized and stored at room temperature or 4°C.

[0056] The composition of a 18 μL reaction solution in a detection system is as follows:

[0057]

[0058] Prepare the reaction solution according to the above proportions, store it in a -20℃ freezer, thaw it on ice before use, and mix it well before use.

[0059] Example 3

[0060] This example demonstrates the application of the reagent kit from Example 2.

[0061] Take 6.4 μL of Candida albicans template nucleic acid, 6.4 μL of Chlamydia trachomatis template nucleic acid, 6.4 μL of Escherichia coli template nucleic acid, 6.4 μL of Group B Streptococcus template nucleic acid, 6.4 μL of Mycoplasma hominis template nucleic acid, and 6.4 μL of Streptococcus pneumoniae template nucleic acid, respectively, mix them with 18 μL of reaction solution, add them to the two sample wells of the chip, seal the sample wells with sealing film, and then load the chip onto the instrument.

[0062] Because this method uses isothermal amplification, it eliminates the need for the temperature-dependent denaturation, annealing, and extension processes required in PCR amplification. The entire reaction is completed under isothermal conditions. The amplification program is set at 63.5℃ for 30 minutes. The centrifugation procedure is as follows: low-speed centrifugation at 1600 rpm for 10 seconds, and high-speed centrifugation at 4600 rpm for 30 seconds.

[0063] The threshold line was set to 800. The instrument's software automatically analyzed the results. When the internal reference showed an amplification curve and Ct < 30, the experimental results were considered valid.

[0064] When the positive control (Ct<30) and the internal standard reaction wells show obvious typical S-shaped amplification curves, and the negative control (Ct<30) and the negative control reaction wells show no amplification curves, but the internal standard reaction wells show obvious typical S-shaped amplification curves, the experiment can be considered successful.

[0065] The microfluidic chip is amplified at a constant temperature on a microfluidic chip detector. The instrument performs real-time fluorescence detection and interprets the results based on the effective amplification curve. If any detection well has a standard S-shaped amplification curve, the well is considered positive, meaning that the sample contains the corresponding bacterial nucleic acid. Detection wells without amplification curves are considered negative, meaning that the sample does not contain the corresponding bacterial nucleic acid.

[0066] If a clear amplification curve appears at the test well within a reaction time of 30 minutes and the Ct value is <30, the test item corresponding to that well is considered positive. If a clear amplification curve appears at the test well, the corresponding test item is considered negative.

[0067] Example 4

[0068] This embodiment verifies the sensitivity and detection limit of the kit described in Example 2.

[0069] Reagent: Reaction solution; 1×10 6 copies / μL, 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0 Plasmids containing gene fragments of Candida albicans, Chlamydia trachomatis, Escherichia coli, Group B Streptococcus, Mycoplasma hominis, and Streptococcus pneumoniae (copies / μL); negative control.

[0070] Following the above detection system, the experimental procedure was performed. The prepared chip was then placed in a constant-temperature amplification instrument for testing. The amplification results are as follows: Figure 2 The limits of detection for primers for Candida albicans (2-A), Chlamydia trachomatis (2-B), Escherichia coli (2-C), Group B Streptococcus (2-D), Mycoplasma hominis (2-E), and Streptococcus pneumoniae (2-F) were 10 mmol / L. 2 copies / μL, 10 1 copies / μL, 1×102 copies / μL, 1×10 2 copies / μL, 1×10 2 copies / μL, 10 1 With a plasmid of copies / μL, Ct < 30, indicating high sensitivity.

[0071] Example 5

[0072] This embodiment is a repeatability verification of the kit described in Example 2.

[0073] Reagents: Reaction solution; 1×10⁻⁶ Candida albicans, Chlamydia trachomatis, Escherichia coli, Group B Streptococcus, Mycoplasma hominis, and Streptococcus pneumoniae 4 Plasmid copies / μL; negative control.

[0074] The experiment was conducted according to the above detection system, and then the chip was placed in the isothermal amplification instrument for experimental detection. Figure 3 This image shows the amplification results of primer repeatability experiments for Candida albicans (3-A), Chlamydia trachomatis (3-B), Escherichia coli (3-C), Group B Streptococcus (3-D), Mycoplasma hominis (3-E), and Streptococcus pneumoniae (3-F). The table below shows the coefficient of variation (CV, %) of the Ct values ​​for primers for Candida albicans, Chlamydia trachomatis, Escherichia coli, Group B Streptococcus, Mycoplasma hominis, and Streptococcus pneumoniae.

[0075]

[0076] Calculations show that

[0077] The coefficient of variation (CV, %) of the Ct value of the primer Candida albicans was 0.92%, which showed good repeatability (less than 5%) and met the requirements.

[0078] The coefficient of variation (CV, %) of the Ct value of the primer Chlamydia trachomatis was 2.05%, which showed good repeatability (less than 5%) and met the requirements.

[0079] The coefficient of variation (CV, %) of the Ct value of the primer Escherichia coli was 3.16%, which showed good repeatability (less than 5%) and met the requirements.

[0080] The coefficient of variation (CV, %) of the Ct value of the streptococci in primer group B was 3.13%, which showed good repeatability (less than 5%) and met the requirements.

[0081] The coefficient of variation (CV, %) of the Ct value of the primer human mycoplasma was 2.02%, which showed good repeatability (less than 5%) and met the requirements.

[0082] The coefficient of variation (CV, %) of the Ct value of the primer Streptococcus pneumoniae was 2.69%, which showed good repeatability (less than 5%) and met the requirements.

[0083] Example 6

[0084] This embodiment is a specificity verification of the kit described in Example 2.

[0085] Reagents: reaction solution; nucleic acid from Candida albicans sample, nucleic acid from Chlamydia trachomatis sample, nucleic acid from Escherichia coli sample, nucleic acid from Group B Streptococcus sample, nucleic acid from Mycoplasma hominis sample, nucleic acid from Streptococcus pneumoniae sample, and negative control.

[0086] The experiment was conducted according to the above detection system, and then the chip was placed in the isothermal amplification instrument for experimental detection. Figure 4 The results of primer-specific amplification experiments for Candida albicans (4-A), Chlamydia trachomatis (4-B), Escherichia coli (4-C), Group B Streptococcus (4-D), Mycoplasma hominis (4-E), and Streptococcus pneumoniae (4-F) show that:

[0087] Except for the positive result of nucleic acid amplification in the Candida albicans sample, no amplification curves were found in the nucleic acid samples of Chlamydia trachomatis, Escherichia coli, Group B Streptococcus, Mycoplasma hominis, Streptococcus pneumoniae, and the negative control. This indicates that the Candida albicans primers can only specifically amplify and detect the nucleic acid of Candida albicans samples, and have good specificity. Generally, there will be no cross-reaction with Chlamydia trachomatis, Escherichia coli, Group B Streptococcus, Mycoplasma hominis, and Streptococcus pneumoniae.

[0088] Except for the positive result of nucleic acid amplification of Chlamydia trachomatis sample, no amplification curves were found for the nucleic acid of Candida albicans sample, Escherichia coli sample, Group B Streptococcus sample, Mycoplasma hominis sample, Streptococcus pneumoniae sample, and the negative control. This indicates that the Chlamydia trachomatis primers can only specifically amplify and detect the nucleic acid of Chlamydia trachomatis sample, and have good specificity. Generally, there will be no cross-reaction with Candida albicans, Escherichia coli, Group B Streptococcus, Mycoplasma hominis, and Streptococcus pneumoniae.

[0089] Except for the positive result of Escherichia coli nucleic acid amplification, no amplification curves were found for Candida albicans, Chlamydia trachomatis, Group B Streptococcus, Mycoplasma hominis, Streptococcus pneumoniae, and the negative control. This indicates that the Escherichia coli primers can only specifically amplify and detect Escherichia coli nucleic acid, and have good specificity. Generally, they will not cross-react with Candida albicans, Chlamydia trachomatis, Group B Streptococcus, Mycoplasma hominis, or Streptococcus pneumoniae.

[0090] Except for the positive result of nucleic acid amplification in the group B streptococcus sample, no amplification curves were found in the nucleic acid samples of Candida albicans, Chlamydia trachomatis, Escherichia coli, Mycoplasma hominis, Streptococcus pneumoniae, and the negative control. This indicates that the group B streptococcus primers can only specifically amplify and detect the nucleic acid of group B streptococcus samples, and have good specificity. Generally, they will not cross-react with Candida albicans, Chlamydia trachomatis, Escherichia coli, Mycoplasma hominis, or Streptococcus pneumoniae.

[0091] Except for the positive result of nucleic acid amplification of Mycoplasma hominis sample, no amplification curves were found for nucleic acid of Candida albicans sample, Chlamydia trachomatis sample, Escherichia coli sample, Group B Streptococcus sample, Streptococcus pneumoniae sample, and the negative control. This indicates that the primers for Mycoplasma hominis can only specifically amplify and detect nucleic acid of Mycoplasma hominis sample, and have good specificity. Generally, there will be no cross-reaction with Candida albicans, Chlamydia trachomatis, Escherichia coli, Group B Streptococcus, and Streptococcus pneumoniae.

[0092] Except for the positive result of Streptococcus pneumoniae nucleic acid amplification, no amplification curves were found for Candida albicans, Chlamydia trachomatis, Escherichia coli, Group B Streptococcus, Mycoplasma hominis, and the negative control. This indicates that the primers for Streptococcus pneumoniae can only specifically amplify and detect Streptococcus pneumoniae nucleic acid, and have good specificity. Generally, they will not cross-react with Candida albicans, Chlamydia trachomatis, Escherichia coli, Group B Streptococcus, or Mycoplasma hominis.

[0093] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A primer composition for the efficient detection of common human pathogens, characterized in that, The primer set includes: The upstream external primer for Candida albicans has the nucleotide sequence shown in SEQ ID NO: 1; The downstream external primer for Candida albicans has the nucleotide sequence shown in SEQ ID NO: 2; The upstream internal primer of Candida albicans has the nucleotide sequence shown in SEQ ID NO: 3; The downstream internal primer of Candida albicans has the nucleotide sequence shown in SEQ ID NO: 4; The upstream loop primer for Candida albicans has the nucleotide sequence shown in SEQ ID NO: 5; The downstream loop primer for Candida albicans has the nucleotide sequence shown in SEQ ID NO: 6; The upstream external primer for Chlamydia trachomatis has the nucleotide sequence shown in SEQ ID NO: 7; The downstream external primer for Chlamydia trachomatis has the nucleotide sequence shown in SEQ ID NO: 8; The nucleotide sequence of the upstream internal primer for Chlamydia trachomatis is shown in SEQ ID NO: 9; The downstream internal primer for Chlamydia trachomatis has the nucleotide sequence shown in SEQ ID NO: 10; The upstream loop primer for Chlamydia trachomatis has the nucleotide sequence shown in SEQ ID NO: 11; The upstream external primer for Escherichia coli has the nucleotide sequence shown in SEQ ID NO: 12; The downstream external primer for Escherichia coli has the nucleotide sequence shown in SEQ ID NO: 13; The upstream internal primer of Escherichia coli has the nucleotide sequence shown in SEQ ID NO: 14; The downstream internal primer of Escherichia coli has the nucleotide sequence shown in SEQ ID NO: 15; The upstream loop primer for Escherichia coli has the nucleotide sequence shown in SEQ ID NO: 16; The downstream loop primer for Escherichia coli has the nucleotide sequence shown in SEQ ID NO: 17; The upstream external primer for Group B Streptococcus is shown in SEQ ID NO: 18; The downstream external primer for Group B Streptococcus is shown in SEQ ID NO: 19; The upstream internal primer for Group B Streptococcus, the nucleotide sequence of which is shown in SEQ ID NO: 20; The downstream internal primer for Group B Streptococcus is shown in SEQ ID NO: 21; The downstream loop primer for Group B Streptococcus is shown in SEQ ID NO: 22; The upstream external primer for Mycoplasma hominis has the nucleotide sequence shown in SEQ ID NO: 23; The downstream external primer for Mycoplasma hominis has the nucleotide sequence shown in SEQ ID NO: 24; The nucleotide sequence of the upstream internal primer for Mycoplasma hominis is shown in SEQ ID NO: 25; The nucleotide sequence of the downstream internal primer for Mycoplasma hominis is shown in SEQ ID NO: 26; The upstream circular primer for Mycoplasma hominis, the nucleotide sequence of which is shown in SEQ ID NO: 27; The upstream external primer for Streptococcus pneumoniae has the nucleotide sequence shown in SEQ ID NO: 28; The downstream external primer for Streptococcus pneumoniae has the nucleotide sequence shown in SEQ ID NO: 29; The upstream internal primer for Streptococcus pneumoniae has the nucleotide sequence shown in SEQ ID NO: 30; The downstream internal primer for Streptococcus pneumoniae has the nucleotide sequence shown in SEQ ID NO: 31; The upstream loop primer for Streptococcus pneumoniae, the nucleotide sequence of which is shown in SEQ ID NO: 32; The downstream loop primer for Streptococcus pneumoniae has the nucleotide sequence shown in SEQ ID NO:

33.

2. The primer composition according to claim 1, characterized in that, The target genes of the primer composition are as follows: The nucleic acid sequence of Candida albicans is U46158, and the target gene is the RSR1 gene; The nucleic acid sequence of Chlamydia trachomatis is HE603228, and the target gene is a partial sequence of the cryptic plasmid gene. The nucleic acid sequence of Escherichia coli is CP054942, and the target gene is the traA gene; The nucleic acid sequence of group B streptococci is CP019814, and the target gene is the cfb gene; The nucleic acid sequence of Mycoplasma hominis is CP055150, and the target gene is referenced from a partial sequence of the alaS gene. The nucleic acid sequence of Streptococcus pneumoniae is MK606437, and the target gene is the cps gene.

3. A kit for the efficient detection of common human pathogens, characterized in that, The invention comprises a microfluidic chip, a reaction solution, and the primer composition as described in claim 1. The microfluidic chip includes four detection regions, each including two sample loading wells and eight detection wells. The eight detection wells are designated as follows: Candida albicans detection well, Chlamydia trachomatis detection well, Escherichia coli detection well, Group B Streptococcus detection well, Mycoplasma hominis detection well, Streptococcus pneumoniae detection well, blank detection well, and internal control detection well. The Candida albicans detection well contains primers for amplifying the Candida albicans sequence; the Chlamydia trachomatis detection well contains primers for amplifying the Chlamydia trachomatis sequence; the Escherichia coli detection well contains primers for amplifying the Escherichia coli sequence; the Group B Streptococcus detection well contains primers for amplifying the Group B Streptococcus sequence; the Mycoplasma hominis detection well contains primers for amplifying the Mycoplasma hominis sequence; the Streptococcus pneumoniae detection well contains primers for amplifying the Streptococcus pneumoniae sequence; the blank detection well serves as a blank control; and the internal control detection well contains internal control primers and an internal control plasmid.

4. The kit according to claim 3, characterized in that, The reaction solution is used in quantities of 18 μL each time, and its composition is as follows: 20mM Tris-HCl 0.2μL 40mM KCl 6.8μL 100mM (NH4)2SO4 1.8μL 80mM MgSO4 1.8μL 1% Tween-20 1.8μL 28mM dNTPs 0.9μL 8000 U / mL Bst enzyme 1.8 μL 1mM SYBR GREEN 0.9μL 4.0×10 -6 1.8 μL of mol / L gold nanoparticles ddH2O 0.2μL.

5. The reagent kit as described in claim 3, characterized in that, The concentration and volume of the primer composition in each detection well of the microfluidic chip are as follows: Candida albicans detection wells: 0.5 μL each of 90 μM Candida albicans upstream external primer and 90 μM Candida albicans downstream external primer, 2 μL each of 180 μM Candida albicans upstream internal primer and 180 μM Candida albicans downstream internal primer, and 1 μL each of 180 μM Candida albicans upstream loop primer and 180 μM Candida albicans downstream loop primer; Chlamydia trachomatis detection wells: 0.5 μL each of 90 μM Chlamydia trachomatis upstream external primer and 90 μM Chlamydia trachomatis downstream external primer, 2 μL each of 180 μM Chlamydia trachomatis upstream internal primer and 180 μM Chlamydia trachomatis downstream internal primer, and 1 μL of 180 μM Chlamydia trachomatis upstream loop primer; Escherichia coli detection wells: 0.5 μL each of 90 μM Escherichia coli upstream external primer and 90 μM Escherichia coli downstream external primer, 2 μL each of 180 μM Escherichia coli upstream internal primer and 180 μM Escherichia coli downstream internal primer, and 1 μL each of 180 μM Escherichia coli upstream loop primer and 180 μM Escherichia coli downstream loop primer; Group B Streptococcus detection wells: 0.5 μL each of 90 μM Group B Streptococcus upstream external primer and 90 μM Group B Streptococcus downstream external primer, 2 μL each of 180 μM Group B Streptococcus upstream internal primer and 180 μM Group B Streptococcus downstream internal primer, and 1 μL of 180 μM Group B Streptococcus downstream loop primer; Mycoplasma hominis detection wells: 0.5 μL each of 90 μM upstream external primer and 90 μM downstream external primer for Mycoplasma hominis, 2 μL each of 180 μM upstream internal primer and 180 μM downstream internal primer for Mycoplasma hominis, and 1 μL of 180 μM upstream loop primer for Mycoplasma hominis; Pneumococcal detection wells: 0.5 μL each of 90 μM upstream external primer and 90 μM downstream external primer for Pneumococcus, 2 μL each of 180 μM upstream internal primer and 180 μM downstream internal primer for Pneumococcus, and 1 μL each of 180 μM upstream loop primer and 180 μM downstream loop primer for Pneumococcus.

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