Composition and detection method for simultaneous detection of eight pathogens including sftsv

Abstract

The present application relates to a method for detecting microbial nucleic acid, in particular, the present application provides a primer and probe composition and a detection method for detecting eight pathogens, including Rickettsia mooseri, Rickettsia typhi, Orientia tsutsugamushi, Ehrlichia, Rickettsia siberica, Anaplasma, Phlebovirus and New Bunyavirus, the primer and probe composition includes eight sets of corresponding primer and probe composition, the nucleotide sequences are shown in SEQ ID NO: 1-35 in the sequence listing, the detection method provided by the present application is optimized under hybridization conditions, eight pathogens can be qualitatively or quantitatively detected in the same reaction system, the whole detection process is simple, rapid, and the minimum detection amount is 10 1 copy / μL viral DNA, with the characteristics of high throughput, low cost, high specificity and high sensitivity.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a primer and probe composition for the combined detection of these eight pathogens, a liquid phase chip, and a non-diagnostic detection method for the liquid phase chip. Background Technology

[0002] In recent years, the situation of tick-borne diseases in my country has become increasingly serious. Most of them are important natural focal diseases and zoonotic diseases. Moreover, tick-borne diseases are caused by a wide variety of pathogens and can carry multiple pathogens at the same time, including viruses, bacteria, rickettsiae and spirochetes.

[0003] Rickettsiae are a major type of pathogens causing tick-borne diseases. Rickettsiae are intracellular parasitic prokaryotic microorganisms, belonging to the order Rickettsiales, including families such as Rickettsiaceae, Anaplasmataceae, and Holosporaceae. The main rickettsiae pathogens affecting humans include genera such as Rickettsiaceae, Orientiaceae, Coxiellaceae, and Ehrlichellaceae, as well as the genus Anaplasmaceae. Rickettsial diseases are prevalent worldwide. In my country, the main types include endemic typhus (Q fever), scrub typhus (also known as smear disease), erythritolosis, spotted fever, and human granulocytic anaplasmosis. The corresponding pathogens are Rickettsia mooseri (MS), Q fever Rickettsia (QF), Orientia tsutsugamushi (Ot), Ehrlichia (Eri), Spotted fever group Rickettsia (SFGR), and Anaplasma phagocytophilum (AP, formerly known as Ehrlichia, Eh).

[0004] Viruses are also one of the main types of pathogens of tick-borne diseases. In recent years, with the deepening of the investigation and research on the spectrum of tick-borne disease viruses, new potential virus spectra have been discovered and identified one after another. For example, Jingmen tick virus (JMTV), which is mainly prevalent in central China, has been detected in human tissue, cattle, tadpoles, and various ticks. However, there are currently no available test reagents or products for the diagnosis and treatment of Jingmen tick virus. Another example is the main pathogen of fever with thrombocytopenia syndrome (SFTSV), also known as neo-SFTSV, which has been highly sporadic in Hubei, Henan, Jiangsu, Zhejiang and other regions since 2004. SFTSV was first isolated and identified in my country in 2011. After infecting humans, the virus causes fever with thrombocytopenia syndrome, fever, thrombocytopenia and leukopenia, and other major clinical manifestations. Severe cases can lead to multiple organ failure and death. SFTSV virus infection cases all have a clear history of tick bites, and its morbidity and mortality rate reaches 12%-30%. Tick-borne encephalitis virus (TBEV), which is mainly prevalent in Northeast China, can cause acute infectious diseases that are common in forest areas from May to July each year, mainly affecting the central nervous system. It is mostly caused by workers being bitten by ticks during their work activities in forest areas, resulting in an acute viral infectious disease of the central nervous system.

[0005] According to literature reports, nearly 40 new tick-borne pathogens have emerged in my country since 1982. Tick-borne diseases are often highly contagious and pathogenic, and most lack effective vaccines. Delayed treatment can lead to serious consequences, posing significant threats to human health and animal husbandry. Mild cases of rickettsia infection primarily present with fever, polymorphic rash, generalized muscle pain, and headache, while severe cases exhibit life-threatening complications such as severe allergic reactions, myocarditis, encephalitis, pulmonary embolism, venous thrombosis, hemorrhage, heart failure, respiratory failure, and septic shock. The aforementioned viruses also present with similar clinical symptoms and epidemiological characteristics, such as fever and leukopenia, making it difficult to identify the specific virus through clinical symptoms and laboratory tests such as blood tests. Currently, most tick-borne pathogen detection methods only target single pathogens, or the sensitivity of methods for combined detection of multiple pathogens is not high enough to detect tick-borne disease infection or epidemics early. Therefore, there is an urgent need for a rapid, simple, accurate, and highly specific product and method that can detect and distinguish pathogens such as rickettsiae and virus types in combination, so as to detect and diagnose early, guide rational clinical drug use as early as possible, or meet the needs of large-scale, high-throughput epidemiological investigation and testing of suspected tick-borne disease clinical samples.

[0006] Multi-Analyte Suspension Array (MASA) technology is a novel biomolecular detection technology that integrates laser technology, flow cytometry, digital signal processing, and traditional chemical techniques. MASA technology supports singleton and multiplex analysis, enabling high-throughput detection of protein and nucleic acid targets across various assay methods. MASA can be widely used in various immunoassays and nucleic acid detections; however, the domestic application of MASA in China is still in its early stages, primarily focused on research and development, and has not yet reached industrialization. Applying MASA to the joint detection of multiple pathogens presents challenges. Since multiplex PCR amplifies multiple pathogens simultaneously within a single reaction system, ensuring that each primer pair does not undergo non-specific amplification with other pathogens is crucial. The presence of multiplex primers and probes, along with the selection of hybridization temperature, are bottlenecks in technology development. Therefore, on the one hand, the primer pairings must be highly specific. On the other hand, primer design must avoid hairpin structures and primer dimers, especially avoiding upstream primers being complementary to any downstream primer for more than four consecutive bases, as this can lead to false positive signals. Finally, establishing a liquid-phase chip detection system is not limited to constructing a good single PCR reaction; the key is how to screen the primer combinations and reaction conditions that are most suitable for multiplex PCR in liquid-phase chips. Summary of the Invention

[0007] The purpose of this invention is to at least partially overcome the deficiencies of the prior art and provide a liquid-phase chip detection method and product that can detect eight tick-borne pathogens in a single tube.

[0008] To achieve the above objectives, this invention provides a primer and probe composition for the joint detection of eight pathogens, including Rickettsia molluscum (MS), Q fever (QF), Orientia tsutsugamushi (Ot), Erickettsia (Eri), Spotted fever group Rickettsia (SFGR), Anaplasma (Eh), Jingmen virus (JMTV), and New Bunyavirus (SFTSV). The primer and probe compositions corresponding to these eight pathogens include, but are not limited to: primer sets MS-rF1, MS-rF2, MS-rR, and probe r-probe, which have identical or complementary nucleotide sequences to the GroEL gene of Rickettsia molluscum; and primer sets MS-rF1, MS-rF2, MS-rR, and probe r-probe, which have identical or complementary nucleotide sequences to the IS1111 gene of Q fever. Primer sets QF-F1, QF-R1, QF-AF, QF-AR and probe QF-probe for complement sequences; primer sets 56KD-F, 56KD-R and probe 56KD-probe for sequences with the same or complementary nucleotide sequences as the htrA gene of 56KD *Orientia tsutsugamushi*; primer sets Eri-F1, Eri-R1, Eri-F2, Eri-R2 and probe E-probe for sequences with the GroEL gene of *Erikis*; primer sets SFGR-F, SFGR-R and probe Rr190-probe for sequences with the 190KD gene of *Rickettsia spectabilis*; and primer sets SFGR-F, SFGR-R and probe Rr190-probe for sequences with the 190KD gene of *Rickettsia spectabilis*; and primers for sequences with the 16S sequence of *Anaplasma tsutsugamushi*. The primer sets Eh-1, Eh-2, Eh-3, Eh-4 and probe H-probe have the same or complementary nucleotide sequences as the rRNA; the primer sets JMTV-F1, JMTV-R1, JMTV-F2, JMTV-R2 and probe S4-probe have the same or complementary nucleotide sequences as the S4 fragment of Jingmen virus; the primer sets F1, R1, F2, R2 and probe M3-probe have the same or complementary nucleotide sequences as the M fragment of neo Bunyavirus; the primers and probes corresponding to these eight pathogens have nucleotide sequences as shown in the sequence listing SEQ ID NO:1-35.

[0009] In a preferred embodiment of the present invention, the probes are prepared into detection microspheres after being labeled with magnetic beads. The probes r-probe, QF-probe, 56KD-probe, E-probe, Rr190-probe, H-probe, S4-probe, and M3-probe are labeled with eight magnetic beads of arbitrary selection and different numbers.

[0010] Preferably, the primer sets corresponding to Q-heat Rickettsia, Ehrlich, Anaplasma, Jingmen virus, and New Bunyavirus are nested PCR primer sets; the primer set corresponding to Mohrlich is an asymmetric semi-nested primer set.

[0011] This invention provides a liquid-phase chip for the joint detection of eight pathogens, comprising standard substances, negative controls, and primer and probe compositions corresponding to the eight pathogens.

[0012] In a preferred embodiment of the present invention, the detection range of the liquid-phase chip for the combined detection of eight pathogens is 1×10⁻⁶. 1 Up to 1×10 6 .

[0013] This invention provides a liquid-phase chip non-diagnostic detection method for the combined detection of eight pathogens, comprising primer and probe compositions corresponding to the eight pathogens involved in the reaction; the detection steps are as follows: detection microspheres: magnetic beads are oscillated and sonicated, and then fully oscillated and coupled with the probes to form detection microspheres; PCR reaction system: a 25 μL reaction system is prepared, including 1.0 μL of DNA, 12.5 μL of PCR premix, 0.5 μL each of forward and reverse primers (10 μM), and deionized water; the DNA in the negative control is replaced with water; PCR amplification reaction: 98℃ pre-denaturation for 5 min, 98℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, 35 cycles, and a final extension at 72℃ for 5 min; hybridization reaction system: 0.5 μL and 33 μL of detection microspheres respectively. 1.5×TMAC, 12μLLTE, 5μL PCR product; Hybridization reaction: denaturation at 95℃ for 5 min, hybridization at 55℃-58℃ for 10-15 min; Detection of microsphere labeling: add 10μL of streptavidin and R-phycoerythrin conjugate (50μg / mL) to the above hybridization solution, and react at 55℃ for 10 min; Result detection: transfer the above reaction solution to a 96-well plate and detect using a liquid chromatography-array suspension analyzer.

[0014] In a preferred embodiment of the present invention, in the liquid-phase chip non-diagnostic detection method for the joint detection of eight pathogens, MagPLEX magnetic beads are selected.

[0015] Preferably, the MagPLEX beads are selected from eight different numbers from the 11 MagPLEX beads numbered 18, 29, 39, 48, 55, 63, 46, 75, 36, 64, and 45.

[0016] In a preferred embodiment of the present invention, in the hybridization reaction of the liquid-phase chip non-diagnostic detection method for the joint detection of eight pathogens, the hybridization temperature is 58°C and the hybridization time is 15 min.

[0017] In a preferred embodiment of the present invention, in the result detection of the liquid-phase chip non-diagnostic detection method for the joint detection of eight pathogens, the liquid-phase chip suspension instrument can detect the fluorescence intensity value of the magnetic beads as the Median value, and output the data as the average fluorescence intensity value of each microsphere, i.e., the MFI value; when the MFI value of the test sample is less than three times the MFI value of the negative control, the output detection result is negative; when the MFI value of the test sample is three times or more the MFI value of the negative control, the output detection result is positive.

[0018] The eight pathogens mentioned in this invention can also be referred to as MS, QF, Ot, Eri, SFGR, Eh, JMTV, and SFTSV, respectively.

[0019] This invention provides a primer and probe composition, liquid phase chip, or non-diagnostic detection method for the joint detection of eight pathogens, specifically for the joint detection of MS, QF, Ot, Eri, SFGR, Eh, JMTV, and SFTSV.

[0020] The beneficial effects of this invention are:

[0021] The liquid-phase chip technology used in this invention is a high-throughput detection technique capable of simultaneously performing quantitative and qualitative analysis on multiple target molecules. For example, this invention can detect eight pathogens (MS, QF, Ot, Eri, SFGR, Eh, JMTV, and SFTSV) using the same sample. Unlike traditional solid-phase chip technology, this invention uses a liquid-phase chip, eliminating the need for sample spotting on the chip. Instead, probes are attached to microsphere carriers and suspended in a liquid environment for sample detection. This method offers advantages such as ease of operation, short processing time, high throughput, and high accuracy, making it suitable for rapid high-throughput detection of large numbers of samples.

[0022] The non-diagnostic detection method for the combined detection of eight pathogens described in this invention can not only detect whether a single pathogen among the eight major tick-borne disease pathogens described in this invention is present in the sample, but also comprehensively detect whether any combination of the eight pathogens described in this invention is present in the sample. Furthermore, it can clearly distinguish between pathogens present in mixed infections in the sample. In this way, a single sample can be used to simultaneously detect eight pathogens, which not only reduces the number of samples used for testing, but also saves time and economic costs. This facilitates doctors in quickly diagnosing the cause of infection for symptomatic treatment, or in discovering the epidemic trend of a certain pathogen, helping disease control departments to take preventive measures as soon as possible. Therefore, the method for the combined detection of eight tick-borne disease pathogens and related detection products described in this invention have good application prospects.

[0023] The liquid-phase chip for the combined detection of eight pathogens described in this invention automatically outputs the detection results via a liquid-phase chip suspension analyzer, eliminating the need for manual interpretation. Compared to traditional multiplex PCR, it not only overcomes the difficulty in distinguishing multiplex amplification products with similar molecular weights through electrophoresis, but also improves the freedom of primer design and selection to a certain extent. The detection results include the average fluorescence intensity, which is statistically significant, and the quantified results can be automatically analyzed, facilitating the automation of detection result reporting.

[0024] This invention successfully established a liquid-phase chip detection method platform capable of jointly detecting eight pathogens, overcoming the bottleneck problem of multiplex PCR primer design, which is of great significance. For routine nucleic acid testing involving simultaneous 8-fold PCR detection, the key and challenging aspect remains the design and optimization of primer combinations. First, the primer and probe combinations for the eight pathogens (MS, QF, Ot, Eri, SFGR, Eh, JMTV, and SFTSV) selected in this invention were obtained through a series of optimizations, including individual amplification screening and multiple combination pairing screening. These combinations exhibit high sensitivity and specificity for both individual and combined detection of these eight pathogens. Second, the primer and probe combinations for these eight pathogens were designed by screening conserved sequences and designing primers targeting specific gene sequences. Furthermore, at least two primer pairs were designed for each pathogen, and for each primer pair obtained through screening and optimization, at least two sets of specific probes were designed. First, pairing was required to avoid any primers or probes for any pathogen having more than four consecutive complementary bases. Second, it was necessary to ensure that there was no interference between the primers and probes for these eight pathogens. The primer / probe combinations must undergo experimental verification through pairing. Verification of the selected primer / probe pairings not only effectively avoids primer dimers but also mitigates false-positive signals caused by interference between the eight groups. Third, through layered optimization and screening of primer / probe pairings, nested PCR primers, and / or semi-nested PCR primers, primer / probe combinations for the eight pathogens described in this invention are obtained. This ensures that the primer / probe combinations for each pathogen exhibit good detection specificity and do not cause non-specific reactions with primers / probes from any of the other seven pathogens. Finally, the primer / probe combinations screened in this invention demonstrate good stability. Even after optimizing and adjusting the reaction system and annealing temperature, the stability of the primer / probe pairings described in this invention remains excellent, exhibiting high independence and specificity. This platform simultaneously detects eight pathogens with high sensitivity and specificity.

[0025] In the process of checking the average fluorescence intensity value of the detection microspheres, this invention, through continuous analysis and verification of the fluorescence intensity value, demonstrates that the liquid-phase chip suspension analyzer can detect the fluorescence intensity value of the magnetic beads as the median value, and outputs the average fluorescence intensity (MFI) value of each type of microsphere, i.e., the MFI value. When the MFI value of the test sample is less than three times that of the negative control, the output test result is negative; when the MFI value of the test sample is three times or more that of the negative control, the output test result is positive. This invention establishes a standard for judging the liquid-phase chip detection results of eight pathogens, facilitating standardized testing and ensuring a more impartial and fair evaluation result when these testing products are used in interlaboratory quality assessments.

[0026] The liquid-phase chip detection method for the combined detection of eight pathogens described in this invention is simple to operate. After PCR amplification, no processing of the PCR product is required; it can be directly used for hybridization with microspheres. The detection steps are simplified, saving purification steps and time, and simplifying the operation. This invention does not require the use of infectious or biosafety-hazardous components during detection, ensuring safety. The detection method is time-efficient (35 minutes operation time, 3 hours total detection time), low-cost, highly specific, and highly sensitive, providing a new technical platform for the combined detection of eight pathogens. Validation with standard substances showed no false positives for two pathogens simultaneously, and the difference between positive and negative values ​​was significant, demonstrating good specificity. Detailed Implementation

[0027] The following detailed description of exemplary embodiments of the present invention, in conjunction with practical applications, illustrates the invention in detail. For ease of explanation, numerous specific details are set forth to provide a comprehensive understanding of the disclosed embodiments. The following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials and reagents used are readily available from conventional commercial sources. Specifically, MagPLEX magnetic beads and the MAGPIX liquid chromatography-suspension array were purchased from Luminex Corporation (USA); EDC solution (10 mg / mL) was purchased from Thermo Fisher Scientific; 0.1 M MES solution, 0.02% Tween-20 solution, 0.1% SDS solution, and pH 8.0 TE solution were all purchased from Sigma-Aldrich; streptavidin-phycoerythrin was purchased from Invitrogen-Thermo Fisher Scientific; and primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. However, it is obvious that one or more embodiments may be implemented without these specific details, and where no specific conditions are specified in the embodiments, they shall be performed under conventional conditions or conditions recommended by the manufacturer.

[0028] Molecular biology experimental methods not specifically described in the examples were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0029] The application scope of this invention only involves biological samples collected from outside the body, referring to tissues, body fluids, or excretions from human or animal bodies. Examples include blood samples (whole blood / serum / plasma) and body fluid samples (urine / feces / cerebrospinal fluid / serous cavity fluid / semen / prostatic fluid / vaginal secretions / gastric juice / duodenal drainage fluid and bile / sputum) that have been separated from a living human or animal body. These are not living human or animal bodies, but rather inanimate biological samples separated from the body. The direct purpose of the detection is not to obtain a diagnosis of a disease or a health status, but merely to conduct laboratory testing or identification of relevant biological information. The results of the test or identification are generally referred to as negative or positive. Furthermore, the testing or identification process only involves the step of obtaining a negative / positive result based on the detected biological information (such as average fluorescence intensity), and does not include the step of directly drawing a disease diagnosis conclusion based on the detected biological information. In other words, the results cannot be directly used for disease diagnosis. Therefore, this invention does not fall under the category of disease diagnosis methods and meets the basic requirements for patent protection subject matter under the Patent Law.

[0030] Example 1: Primer and Probe Design

[0031] This invention provides a liquid-phase chip detection method capable of jointly detecting six rickettsiae and two viruses among tick-borne disease pathogens. First, the target sequences for the primers and probes are: the GroEL gene of Rickettsia moraceae (MS), the IS1111 gene of Q fever (QF), the 56-kDa htrA gene of Orientia tsutsugamushi (Ot), the GroEL gene of Eri, the 190kD protein gene of SFGR, the 16S rRNA gene of Anaplasma (Eh), the S4 fragment of JMTV virus, and the M fragment of SFTSV. Based on the premise that at least two sets of primers can be optimized and screened for each pathogen, primer design software was used to analyze the annealing temperature values ​​of primers and probes and the probability of dimer formation between each primer and probe. Finally, at least two sets of primers were optimized and screened for each pathogen, meaning each primer set corresponds to at least two probes. Secondly, the screened primers and probes were tested to verify their sensitivity and specificity against a single pathogen. Specific experiments were conducted to verify the amplification effect of primers corresponding to a single pathogen and the detection effect of primer-probe pairing. Thirdly, the detection performance of a single-tube mixed detection of primers and probes for multiple pathogens was verified after mixing the optimized primers and probes for each pathogen. Finally, a primer-probe combination that can specifically and sensitively detect each pathogen without interference was screened. The sequences of the screened primer and probe combinations are detailed in Table 1.

[0032] Table 1 Sequence List

[0033]

[0034] The inventors of this application conducted extensive research (designing three sets of primers and two corresponding probe sequences for each pathogen, failing many times, and finally unexpectedly screened the primer and probe composition described in this invention that can specifically amplify plasmid DNA containing Rickettsia molluscum (MS), Q fever (QF), Orientia scrub typhus (Ot), Erickettsia (Eri), Spotted fever group Rickettsia (SFGR), Anaplasma (Eh), Jingmen virus (JMTV), and New Bunyavirus (SFTSV).

[0035] Example 2: Establishment and optimization of a combined detection method for eight pathogens

[0036] 2.1 Preparation of Standard Reference Materials

[0037] The gene fragments related to the eight pathogens of this invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Recombinant plasmids were obtained by cloning the target gene fragments of the pathogens. After plasmid extraction, the concentration was determined using a NAS-99 micro spectrophotometer. The concentration was converted to copy number based on the Avogadro number, and the copy number (copies / μL) was 6.02 × 10⁻⁶. 23 × Measured concentration (ng / μl×10) -9 (DNA length × 660) = copies / μl, then serially diluted with nuclease-free water to 1 × 10⁻⁶. 6 1×10 5 1×10 4 1×10 3 1×10 2 1×10 1 Copy / μl, aliquot and store at -20℃ for later use.

[0038] 2.2 Preparation of detection microspheres

[0039] 1. Remove the magnetic beads (MagPLEX magnetic beads, Luminex Corporation) stock solution and allow it to return to room temperature. In this invention, the magnetic beads are selected from 11 different numbers: 18, 29, 39, 48, 55, 63, 46, 75, 36, 64, and 45. In this embodiment, the magnetic beads selected are numbered 18, 29, 36, 48, 55, 63, 46, and 64. Each magnetic bead is vortexed for 1 minute and then sonicated. 50 μL of each magnetic bead is then transferred to a centrifuge tube, the centrifuge tube is placed on a magnetic rack for 1 minute, and the supernatant is discarded.

[0040] ② Add 50 μL of 0.1 M MES (pH 4.5, purchased from Sigma) to resuspend the contents and sonicate for 20 seconds.

[0041] ③ In this invention, probes corresponding to eight pathogens are coupled to eight magnetic beads with different numbers. 1 μL of 100 μM probes corresponding to the eight pathogens (i.e., a final concentration of 20 pmol / μL) is added to each of the eight magnetic beads with different numbers, and the mixture is shaken to mix.

[0042] ④ Prepare fresh EDC solution (10 mg / mL, purchased from Thermo Fisher Scientific) using ddH2O, add 2.5 μL of 10 mg / mL EDC solution, shake to mix, and incubate at room temperature in the dark for 30 min;

[0043] ⑤ Prepare a fresh EDC solution (10 mg / mL) using ddH2O, add 2.5 μL of the 10 mg / mL EDC solution, shake to mix, and incubate at room temperature in the dark for 30 min.

[0044] ⑥ Add 1 mL of 0.02% Tween-20 (purchased from Sigma) to each centrifuge tube, shake to mix; place the centrifuge tube on a magnetic rack for 1 min, and discard the supernatant;

[0045] ⑦ Add 1 mL of 0.1% SDS (purchased from Sigma) to resuspend the contents, shake to mix; place the centrifuge tubes on a magnetic rack for 1 min, and discard the supernatant;

[0046] ⑧ Add 100 μL of TE (pH 8.0, purchased from Sigma) to resuspend the microspheres, oscillate and sonicate for about 20 seconds to obtain detection microspheres (i.e., coupled microspheres formed by probes labeled with eight different magnetic beads) for the joint detection of eight pathogens such as SFTSV. Store at 4°C in the dark for later use.

[0047] 2.3 Reaction System and Reaction Amplification

[0048] PCR reaction system (25 μL): 12.5 μL PCR premix (Multiplex PCR Plus kit, purchased from Qiagen), 0.50 μL each of forward and reverse primers (10 μM) (i.e., 1 μL of primer to a final concentration of 20 pmol / μL), 1.0 μL DNA (standard substance to a final concentration of 1 × 10⁻⁶). 2 Add deionized water to make up to 25 μL.

[0049] PCR amplification reaction: 98℃ pre-denaturation for 5 min, 98℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, 35 cycles, and a final extension at 72℃ for 5 min.

[0050] 2.4 Hybridization steps

[0051] ① Use an eddy current oscillator to fully vibrate the coupled microspheres;

[0052] ② Amount used in one reaction system: Add 0.5 μL of each type of microsphere to a PCR tube, add 33 μL of 1.5×TMAC and 12 μL of LTE (pH 8.0), mix thoroughly, and add 5 μL of the PCR product from the multiplex reaction;

[0053] ③ Denature at 95℃ for 5 minutes, and select a certain hybridization temperature and a certain hybridization time;

[0054] ④ Add 10 μL of 50 μg / mL streptavidin and R-phycoerythrin conjugate to each PCR tube, react at 55℃ for 10 min, then transfer to a 96-well plate and use a liquid chromatography-array suspension analyzer for detection;

[0055] ⑤ Prepare the instrument by turning it on according to the instrument operation instructions, and place the 96-well plate into the detection chamber for testing;

[0056] 2.5 Result Judgment

[0057] Hybridization detection was performed using a negative control (B) as the background. For each detection system and background, the instrument output data was the average fluorescence intensity value of each type of microsphere in the corresponding reaction system (i.e., the system automatically detected 50 microspheres of each type in each system, and the average fluorescence intensity value of the microspheres automatically displayed by the system was the average of the fluorescence intensity values ​​of the 50 microspheres), namely the MFI value, and the background (negative control: water) fluorescence intensity value, denoted as the BFI value. When the MFI value of the sample to be tested was more than three times the background signal intensity of this detection, it was judged as positive (+), otherwise it was judged as negative (-), that is, MFI(+)≥3×BFI, MFI(-)<3×BFI.

[0058] 2.6 Optimization of hybridization conditions

[0059] In step ③ of the hybridization process described above, the hybridization temperature and time are adjusted to further optimize the hybridization conditions.

[0060] Optimization of hybridization temperature: The hybridization time was initially fixed at 10 min. Based on the range of Tm values ​​in the primer and probe combinations, 55℃ and 58℃ were selected as parallel experiments for hybridization temperature verification. The PCR products of the mixed template were used as the test samples, and the MFI value was used to interpret the results. The results are shown in Table 2 below:

[0061] Table 2 Results of parallel hybridization temperature experiments

[0062]

[0063] Note: B in the table represents Blank, which is the negative control (water) in the PCR reaction; the test results are expressed as MFI values ​​and / or "+" / "-".

[0064] Comparing the test results in Table 2, the final concentration of the samples (standard substances) was 1×10⁻⁶. 2 At a visible temperature of 55℃, the detection sensitivity of erythropoietin and anaplasm was low (MFI values ​​of 562 and 568, respectively), resulting in false negatives and unclear positive / negative differentiation, i.e., the presence of detection grayscale. However, at a temperature of 58℃, the detection sensitivity significantly improved, the difference between positive and negative values ​​was obvious, and there were no false negatives, i.e., no detection grayscale. Therefore, the hybridization temperature with better specificity and sensitivity for this detection method was ultimately determined to be 58℃.

[0065] Optimization of hybridization time: After determining the optimal hybridization temperature as 58℃ in the previous step, parallel experiments were conducted with two hybridization times (10 min and 15 min) for verification. The PCR products of the mixed template were used as the test samples to compare the effects of different hybridization times on signal intensity (e.g., MFI value). The results are shown in Table 3 below:

[0066] Table 3 Comparison of detection results at different hybridization times

[0067]

[0068] Note: B in the table represents Blank, which is the negative control (water) in the PCR reaction; the test results are expressed as MFI values ​​and / or "+" / "-".

[0069] As can be seen from the comparison of the results at different hybridization times in Table 3, the final concentration of the sample (standard substance) was 1×10⁻⁶. 2 When the hybridization time was 10 min, there were non-specific reactions between Rickettsia molluscum and Orientia scrub typhus, and between Rickettsia spectabilis and Orientia scrub typhus (MFI values ​​were 815 and 970, respectively), showing weak positive results. When the hybridization time was 15 min, the difference between negative and positive MFI values ​​was significant, and no weak positive results were observed. Therefore, a hybridization time of 15 min is relatively more advantageous.

[0070] Therefore, the final optimized hybridization conditions are: the hybridization reaction time is 5 min of pre-denaturation at 95℃, and the hybridization reaction time is preferably 15 min at a hybridization temperature of 58℃.

[0071] Example 3: Performance Validation of the Combined Detection Method for Eight Pathogens

[0072] 3.1 Specificity

[0073] The specificity of the combined detection method for eight pathogens was validated using the optimized PCR reaction system and hybridization conditions. The specific reaction conditions were as follows: PCR reaction system (25 μL): 12.5 μL PCR premix (Multiplex PCR Plus kit, purchased from Qiagen), 0.50 μL each of forward and reverse primers (10 μM) (i.e., 1 μL primer to a final concentration of 20 pmol / μL), 1.0 μL DNA (standard substance to a final concentration of 1 × 10⁻⁶). 3 Add deionized water to a final volume of 25 μL. PCR amplification reaction: 98℃ pre-denaturation for 5 min, 98℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, 35 cycles, with a final extension at 72℃ for 5 min. Hybridization conditions: 95℃ pre-denaturation for 5 min, 58℃ hybridization for 15 min.

[0074] The final concentration of the eight standard substances selected was 1×10⁸.3 The specificity of the combined detection method for eight pathogens against the standard substance was evaluated. The results are shown in Table 4 below.

[0075] Table 4 Specific detection results

[0076]

[0077] Note: B in the table represents Blank, which is the negative control (water) in the PCR reaction; the test results are expressed as MFI values ​​and / or "+" / "-".

[0078] As shown in Table 4: Compared with Tables 2 and 3, when the concentration of the test sample (standard substance) (from 1×10⁻⁶) decreased, the concentration of the test sample (standard substance) decreased. 2 →1×10 3 The MFI (Mean Filtration Index) value increased significantly, and the background level (BFI) in the detection value (i.e., MFI value) also increased. Along with the increase in the MFI value of positive samples, the difference between positive and negative values ​​became exceptionally pronounced. Furthermore, during the detection of mixed PCR products, no two pathogens were simultaneously positive. For example, when the novel Bunyavirus was positive, the other seven pathogens were all negative. There were no non-specific false positive results; that is, no two pathogens were positive simultaneously, and the specificity verification results for the other seven pathogens were the same. Therefore, the combined detection method for the eight pathogens described in this invention has good specificity.

[0079] 3.2 Sensitivity

[0080] The sensitivity of the combined detection method for eight pathogens described in this invention was verified based on the PCR reaction and hybridization steps of the above-mentioned specificity verification experiment. Six concentration gradients of standard substances were prepared: non-pathogen plasmids were used for detection, and the concentrations were measured using a NAS-99 micro spectrophotometer and calculated according to the formula: Copy number (copy / μL) = (6.02 × 10⁻⁶). 23 × Measured concentration (ng / μl×10) －9 The copy number of the plasmid was calculated by dividing the plasmid length by 660 (DNA length × 660) by the number of copies per μl. Then, the plasmid was serially diluted to 1 × 10⁻⁶ using nuclease-free water. 6 Copy / μl, 1×10 5 Copy / μl, 1×10 4 Copy / μl, 1×10 3 Copy / μl, 1×10 2 Copy / μl, 1×10 1 Copy / μl, aliquot and store at -20℃ for later use. In this example, PCR amplification and subsequent hybridization steps were performed on the standard substances at six concentration gradients. The six concentrations are abbreviated as: S1 = 1 × 10⁻⁶. 1Copy / μl, S2 = 1 × 10 2 Copy / μl, S3 = 1 × 10 3 Copy / μl, S4 = 1 × 10 4 Copy / μl, S5 = 1 × 10 5 Copy / μl, S6 = 1 × 10 6 The sensitivity of the detection results corresponding to different concentrations of standard substances was compared using copies / μl. The results are shown in Table 5 below.

[0081] Table 5. Detection sensitivity of standard substances at different concentrations

[0082]

[0083]

[0084] Note: In the table, B represents Blank, i.e., the negative control (water) in the PCR reaction; S1-S6 represent the concentrations of the standard substances, respectively: 1×10⁻⁶. 1 1×10 2 1×10 3 1×10 4 1×10 5 1×10 6 The concentration unit is copies / μL; positive results are expressed as MFI values, and negative results are expressed as "-".

[0085] As shown in Table 5 above: all standard substances containing eight pathogen plasmids, 1×10 1 Up to 1×10 6 All of them can be detected positively, and the difference between positive and negative results is significant. Therefore, the sensitivity of the liquid phase chip and detection platform for the combined detection of eight pathogens described in this invention can reach 10. 1 Compared with existing multiplex PCR detection methods (10 3 It improves sensitivity by at least 100 times and has good sensitivity.

[0086] The inventors of this application, after extensive research and numerous failures, finally screened the primer and probe composition described in this invention, which can specifically amplify DNA from eight plasmids: MS, QF, Ot, Eri, SFGR, Eh, JMTV, and SFTSV. In the development of multiplex detection, primer design and validation are crucial. Non-specific amplification bands frequently appear; sometimes non-specific amplification exists even in individual detections, while in other cases, non-specific amplification interference only occurs during multiplex detection when individual amplification is normal. This invention aims to detect eight tick-borne pathogens simultaneously. However, using any single primer pair for MS, QF, Eri, Eh, JMTV, and SFTSV resulted in significant non-specific amplification interference. Later, a nested PCR method using two primer pairs in a single tube was developed to address the mutual interference and non-specificity issues in multiplex detection of these six pathogens. The positive result was a reduction in non-specific amplification of QF, Eri, Eh, JMTV, and SFTSV; however, insufficient detection sensitivity still existed for MS. Further adjustments and verifications were made for the joint detection of MS. Primer pairings were optimized, and primers screened using a semi-nested PCR method were unexpectedly used, solving the sensitivity problem in MS. Experimental verification showed that using semi-nested PCR for MS not only improved sensitivity and specificity in joint detection but also avoided interference with the other seven pathogens. Furthermore, the inventors will continue to conduct in-depth research on joint detection methods for multiple pathogens, breaking through the technical bottlenecks of cross-interference, insufficient sensitivity, and insufficient specificity between certain pathogens (such as novel Bunyavirus and tick-borne encephalitis virus) in the joint detection of more than eight pathogens.

[0087] Ultimately, this invention provides a method for the combined detection of eight pathogens. Based on the overall concept of this invention, eight primer and probe combinations were successfully screened, and the hybridization temperature and time were optimized. The method is simple to operate and time-saving. It not only enables the combined detection of eight pathogens but also distinguishes between the positive results of the eight pathogens, achieving a sensitivity of up to 10. 1 Furthermore, there is no cross-influence, and the specificity is good; furthermore, the present invention does not require processing of PCR products, and the detection steps are more simplified; no infectious or biosafety-hazardous components are used in the detection process, making it safe to use; the present invention establishes the criteria for determining positive and negative results, and the data output can be digitized and automated, showing good application prospects.

[0088] The specific embodiments of the present invention have been described in detail, making them readily understandable and implementable by those skilled in the art. However, based on all the disclosed descriptions, various modifications or substitutions can be made to those details—changes that will be obvious to those skilled in the art—and these changes can be implemented in other embodiments. The invention should not be limited to the embodiments shown herein, and all such changes should be within the scope of protection of the present invention. Therefore, the full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A primer and probe composition for the combined detection of eight pathogens, said eight pathogens including Rickettsia molluscum, Rickettsia Q fever, Orientia scrub typhus, Ehrlich, Rickettsia spectabilis, Anaplasma, Jingmen virus, and Novo Bunyavirus; The primer and probe composition includes, but is not limited to: Primer set MS-rF1, MS-rF2, MS-rR and probe r-probe with nucleotide sequences identical or complementary to those of the GroEL gene of Rickettsia molluscum; Primer sets QF-F1, QF-R1, QF-AF, QF-AR and probe QF-probe that have the same or complementary nucleotide sequences as the IS1111 gene of Q thermorickettsia; Primer set 56KD-F, 56KD-R and probe 56KD-probe with nucleotide sequences identical or complementary to the htrA gene of 56KD of Orientia tsutsugamushi. Primer set Eri-F1, Eri-R1, Eri-F2, Eri-R2 and probe E-probe with nucleotide sequences that are identical or complementary to the GroEL gene of Ehrlich; Primer set SFGR-F, SFGR-R and probe Rr190-probe with nucleotide sequences that are identical or complementary to the 190KD gene of spotted heat group Rickettsia; Primer set Eh-1, Eh-2, Eh-3, Eh-4 and probe H-probe that have the same or complementary nucleotide sequence as the 16S rRNA of anaplasma; Primer set JMTV-F1, JMTV-R1, JMTV-F2, JMTV-R2 and probe S4-probe with nucleotide sequences that are identical or complementary to the S4 fragment of Jingmen virus; Primer set F1, R1, F2, R2 and probe M3-probe with nucleotide sequences identical or complementary to the M fragment of the new Bunyavirus; The primer sets corresponding to the Q-heat Rickettsia, Ehrlich, Anaplasma, Jingmen virus, and Novo Bunyavirus constitute nested PCR primer sets; the primer set corresponding to the Morohrichthys is an asymmetric semi-nested primer set. The nucleotide sequences of MS-rF1, MS-rF2, MS-rR, r-probe, QF-F1, QF-R1, QF-AF, QF-AR, QF-probe, 56KD-F, 56KD-R, 56KD-probe, Eri-F1, Eri-R1, Eri-F2, Eri-R2, E-probe, SFGR-F, SFGR-R, Rr190-probe, Eh-1, Eh-2, Eh-3, Eh-4, H-probe, JMTV-F1, JMTV-R1, JMTV-F2, JMTV-R2, S4-probe, F1, R1, F2, R2, and M3-probe are shown in the sequence listing SEQ ID NO:1-35.

2. The primer and probe composition according to claim 1, characterized in that: The probes were prepared into detection microspheres after being labeled with magnetic beads. The probes r-probe, QF-probe, 56KD-probe, E-probe, Rr190-probe, H-probe, S4-probe, and M3-probe were labeled with eight magnetic beads of arbitrary numbers.

3. A liquid phase chip for the combined detection of eight pathogens, characterized in that: It comprises a standard substance, a negative control, and the primer and probe composition as described in any one of claims 1-2.

4. The liquid phase chip for joint detection of eight pathogens according to claim 3, characterized in that: The detection range of the liquid phase chip is 1×10⁻⁶. 1 Copy / μl to 1×10 6 Copy / μL.

5. A liquid-phase chip non-diagnostic detection method for the combined detection of eight pathogens, characterized in that: The method comprises the primer and probe composition as described in any one of claims 1-2 participating in the reaction; the detection step is as follows: Detection microspheres: After the magnetic beads are vibrated and sonicated, they are fully vibrated and coupled with the probe to form detection microspheres; PCR reaction system: Prepare a 25 μL reaction system, including 1.0 μL DNA, 12.5 μL PCR premix, 1 μL primers including 0.5 μL each of 10 μM forward and reverse primers, and deionized water; the DNA in the negative control is replaced with water; wherein, the final concentration of primers is 20 pmol / μL; PCR amplification reaction: 98℃ pre-denaturation for 5 min, 98℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, 35 cycles, and a final extension at 72℃ for 5 min; Hybridization reaction system: 0.5 μL of detection microspheres, 33 μL of 1.5×TMAC, 12 μL of LTE, and 5 μL of PCR product; Hybridization reaction: denaturation at 95℃ for 5 min, hybridization at 55℃-58℃ for 10-15 min; Detection of microsphere labeling: Add 10 μL of streptavidin and R-phycoerythrin conjugate (50 μg / mL) to the above hybridization solution and react at 55℃ for 10 min; Results detection: The above reaction solution was transferred to a 96-well plate and detected using a liquid chromatography-in-chip suspension analyzer.

6. The liquid-phase chip non-diagnostic detection method for the combined detection of eight pathogens according to claim 5, characterized in that: The magnetic beads used are MagPLEX magnetic beads.

7. The liquid-phase chip non-diagnostic detection method for the combined detection of eight pathogens according to claim 5, characterized in that: In the hybridization reaction, the hybridization temperature was 58°C and the hybridization time was 15 min.

8. The liquid-phase chip non-diagnostic detection method for the combined detection of eight pathogens according to claim 5, characterized in that: In the result detection, the liquid phase chip suspension instrument can detect the fluorescence intensity value of the magnetic beads as the Median value, and output the average fluorescence intensity value of each microsphere, i.e., the MFI value. When the MFI value of the test sample is less than three times the MFI value of the negative control, the output test result is negative; when the MFI value of the test sample is three times or more the MFI value of the negative control, the output test result is positive.

9. The application of the primer and probe composition as described in any one of claims 1-2, or the liquid phase chip for the combined detection of eight pathogens as described in any one of claims 3-4, or the liquid phase chip for the combined detection of eight pathogens as described in any one of claims 5-8, in the combined non-diagnostic detection of eight pathogens: Rickettsia molluscum, Rickettsia Q fever, Orientia scrub typhus, Ehrlich, Rickettsia spectabilis, Anaplasma, Jingmen virus, and Novo Bunyavirus.

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