SFTSV, SFGR and Ot triple droplet type digital PCR detection method

Abstract

A triple droplet type digital PCR (ddPCR) detection method for the severe fever with thrombocytopenia syndrome virus (SFTSV), spotted heat group rickettsia (SFGR) and tsutsugamushi Ot is established. According to the method, specific primers and probes are designed based on an SFTSV L gene, an SFGR 17Kd gene and an Ot 47Kd htrA gene, the reaction temperature and the concentration of the primers and probes are optimized to establish an SFTSV, SFGR and Ot triple ddPCR reaction system, and the specificity, sensitivity and repeatability of the triple ddPCR reaction system are analyzed. According to results, the optimal primer concentration and probe concentration of triple digital PCR are 600nmol / L and 250nmol / L respectively, and the optimal annealing temperature is 60 DEG C; the correlation coefficients of the triple ddPCR SFTSV, the triple ddPCR SFGR and the triple ddPCR Ot standard curve are 0.98, 0.99 and 0.98 respectively, and the lowest detection lower limits are 0.59 copies / mu L, 0.46 copies / mu L and 0.36 copies / mu L respectively. According to the invention, the SFTSV, SFGR and Ot triple digital PCR detection method is established, the sensitivity is high, the specificity is strong, and quantitative detection of three pathogens in SFTS suspected cases can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology and relates to a triple droplet digital PCR detection method for SFTSV, SFGR and Ot and a kit used therefor. Background Art

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease, and its pathogen is severe fever with thrombocytopenia syndrome virus (SFTSV). Tick-borne spotted fever is caused by infection with spotted fever group rickettsia (SFGR). Scrub typhus is a natural zoonotic disease caused by infection with Orientia tsutsugamushi (Ot), and the disease mainly occurs through bites of chiggers. The early clinical symptoms of the three diseases are relatively similar, and the distribution areas and epidemic seasons of the cases have a certain overlap. The epidemiological characteristics of the cases, such as outdoor activity history, history of raising or contacting wild animals, history of tick or chigger bites, etc., are basically the same, making it difficult to differentially diagnose the three diseases. Du Juan et al. detected the presence of various spotted fever rickettsiae, including Rickettsia raoultti and Rickettsia japonica, in suspected SFTS cases in Xinyang, Henan and Tai'an, Shandong. The results of many studies show that there are certain misdiagnosis and missed diagnosis phenomena among SFTS, spotted fever and scrub typhus, and there are also cases of co-infection. Since there are certain differences in the diagnosis and treatment plans for the three diseases, some literature shows that misdiagnosis of the above patients may lead to death of the cases.

[0003] At present, the diagnosis of SFTS, spotted fever, and scrub typhus mainly relies on laboratory testing. Molecular biology detection techniques mainly detect some genes of SFTSV, SFGR, and Ot through methods such as nested PCR, real-time quantitative fluorescence PCR, and loop-mediated isothermal amplification (Lamp). At the same time, the vast majority of the above molecular biology detection methods are qualitative detection methods. Although qPCR can be used for quantitative detection, it depends on the standard curve of known samples. Droplet Digital PCR (ddPCR) is a new type of PCR detection technology developed in recent years and is the third-generation PCR technology developed by combining fluorescence PCR technology on the basis of traditional PCR. In ddPCR, the DNA / RNA molecules to be detected are diluted to the single-molecule level by the reaction system with fluorescent probes and randomly encapsulated in droplets for reaction. According to the principle of Poisson distribution, the absolute quantification of the sample to be detected is achieved by reading and counting the detected fluorescent units. This quantification method does not depend on the standard curve and has good sensitivity, specificity, and high tolerance to PCR inhibitors. At present, various pathogen detection methods based on ddPCR have been established at home and abroad, such as novel coronavirus, Japanese encephalitis virus, West Nile virus, etc. Summary of the Invention

[0004] The purpose of the present invention is to provide a triple droplet digital PCR detection method for SFTSV, SFGR, and Ot and a kit used therefor.

[0005] To achieve the above object and other related objects, the technical solution provided by the present invention is: A kit for triple droplet digital PCR detection of SFTSV, SFGR, and Ot, comprising the following primers and probes:

[0006] SFTSV-L4919-38-F: 5’-CATACACWGAGGAGTACAAG-3’;

[0007] SFTSV-L5004-24R: 5’-CAYARRGTCATGGTCCTWGAT-3’;

[0008] Probe SFTSV-L4982P: ROX-5’-TCCTRATGACCTCTGATGGYT-3’;

[0009] 17Kd-F: 5’-GGTTCTCAATTCGGTAAG-3’;

[0010] 17Kd-R: 5’-TATCCTGCTCATCCATAC-3’;

[0011] Probe 17Kd-P: FAM-5’-TCCACCAAGAACTGCTCCAAG-3’;

[0012] htrA-670F: 5’-GTATCTTACTCAGGCATAA-3’;

[0013] htrA759R: 5’-AGCATAACATTTAACATACCA-3’;

[0014] Probe htrA699P: VIC-5’-TCCATCTAATACTGTACTTGAAGCA-3’;

[0015] Plasmids pUC57_SFTSV, pUC57_SFGR and pUC57_Ot.

[0016] To achieve the above and other related objectives, the technical solution provided by the present invention is: A triple droplet digital PCR detection method for SFTSV, SFGR and Ot, comprising the following steps:

[0017] Step 1: Single digital PCR amplification

[0018] 3 μL of PCR buffer, 1.35 μL each of 10 μM upstream and downstream primers, 0.375 μL of 10 μM probe, 2 μL of plasmid as DNA template, and made up to 15 μL with ddH2O; The reaction conditions are: pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 s, annealing at 60 or 61 °C for 1 min, 40 cycles;

[0019] Step 2: Establishment and optimization of multiplex digital PCR

[0020] (1) Temperature verification: 3 μL of PCR buffer, 1.35 μL each of 10 μM primers, 0.375 μL of 10 μM probe, 2 μL of DNA template, and made up to 15 μL with ddH2O; The annealing temperature is set at 59 - 62 °C; The reaction conditions are pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 s, annealing at 59 - 62 °C for 1 min, 40 cycles;

[0021] (2) Sensitivity experiment and repeatability experiment

[0022] To verify the sensitivity of the established triple ddPCR for detecting SFTSV, SFGR and Ot, 10 μL each of the dissolved positive standards pUC57_SFTSV, pUC57_SFGR and pUC57_Ot were taken, mixed with 70 μL of H2O, and then serially diluted 10-fold. Select 10 -6 ,10 -7 ,10 -8 ,10 -9 ,10-10 ,10 -11 Plasmids with different dilutions were subjected to multiplex ddPCR detection, with each gradient repeated 3 times. A standard curve was plotted using GraphPad Prism 9, and the coefficient of variation for each gradient was calculated.

[0023] (3) Threshold determination

[0024] The established triple ddPCR was applied to detect 30 negative samples, 15 SFTSV-positive specimens, 8 SFGR-positive samples, and 2 Ot-positive samples, and the optimal cut-off value was calculated using MedCalc software.

[0025] (4) Specificity of triple ddPCR

[0026] The established ddPCR reaction system was used to detect the specificity of triple ddPCR for nucleic acid templates of hemorrhagic fever with renal syndrome virus, dengue virus, Brucella, and Streptococcus suis.

[0027] Due to the application of the above technical solution, the advantages of the present invention compared with the prior art are:

[0028] Based on droplet digital PCR, the present invention establishes a multiplex ddPCR technology for three pathogens, SFTSV, SFGR, and Ot, which can quantify the pathogen content while detecting the pathogens, enabling early screening of the pathogens, timely detection of infected individuals, quantification of the pathogen load in the infected individuals, prediction of the disease process of the patients, and providing a reference basis for effectively controlling the development of the disease process. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a one-dimensional diagram of singleplex ddPCR detection for SFTSV, SFGR, and Ot. A: SFTSV positive plasmid standard (P1), yellow droplets; B: SFGR positive plasmid standard (P2), blue droplets (P3); C: Ot positive plasmid standard, green droplets; N blank control, gray droplets.

[0030] Figure 2 It is the optimization of digital PCR temperature; A: Optimization of annealing temperature for SFTSV; B: Optimization of annealing temperature for SFGR; C: Optimization of annealing temperature for Ot.

[0031] Figure 3 It is the optimization of digital PCR primer and probe concentrations; A - C: Graphs for screening digital PCR primer concentrations; D - F: Graphs for screening digital PCR probe concentrations.

[0032] Figure 4 It is the standard curve of sensitivity for SFTSV.

[0033] Figure 5It is the sensitivity standard curve graph of SFGR.

[0034] Figure 6 It is the sensitivity standard curve graph of Ot. Specific implementation manners

[0035] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this embodiment.

[0036] Please refer to Figures 1-6 Note that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those skilled in the art to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they do not have a substantial technical meaning. Any modification of the structure, change of the proportional relationship or adjustment of the size. The following embodiments are provided to better understand the present invention, rather than to limit the present invention. The experimental materials used in the following embodiments are all obtained from conventional biochemical reagent stores without special instructions.

[0037] Example 1: Triple droplet digital PCR detection method for SFTSV, SFGR and Ot and kit used therefor

[0038] Materials and methods

[0039] Reagents and instruments

[0040] CqEx-DNA / RNA Virus Nucleic Acid Extraction and Purification Kit (Xi'an, China, Tianlong), AD16 Digital PCR Instrument (Hangzhou, China, Pilot); Q5 Fluorescent PCR Instrument; Pilot Digital PCR Universal Kit (including microfluidic chip); TaKaRa PrimeScriptTM Ⅳ 1st strand cDNA Synthesis Mix; TaKaRa Premix Ex TaqTM (Probe qPCR); Qiagen Blood DNA Extraction Kit.

[0041] Clinical samples SFTS suspected clinical samples are from SFTS suspected case samples collected in Chuzhou area in 2024; healthy population specimens are from Huaiyuan area in Anhui; SFGR and Ot clinical samples are from case samples in Anqing City, Anhui; serum specimens are extracted for RNA according to the instructions of Tianlong automatic nucleic acid extractor, and cDNA specimens are obtained by reverse transcription according to the instructions of the reverse transcription kit. Whole blood specimens are extracted for DNA according to the instructions of Qiagen Blood DNA Extraction Kit. The obtained cDNA and DNA specimens are stored at -80 °C for later use.

[0042] Primer probe and plasmid synthesis

[0043] Download the representative sequences of the SFTSV L gene, the 17Kd gene of Rickettsia spotted fever, and each genotype of Orientia tsutsugamushi htrA from the NCBI database. Use the Megalign software for alignment and analysis, screen for highly conserved sequences, design probe primers using the Beacon Designer 7 software, import the designed probe primers into NCBI's Primer-Blast to verify primer specificity, select appropriate primer probes, and synthesize them by Wuhan Tianyi Huayu Gene Technology Co., Ltd., as shown in Table 1.

[0044] Table 1 Primer and probe sequences

[0045]

[0046] Using the 3 pairs of primers in Table 1 as the target, commission Wuhan Tianyi Huayu Gene Technology Co., Ltd. to synthesize plasmid standards pUC57_SFTSV, pUC57_SFGR, and pUC57_Ot with PUC57 as the vector. Dissolve each tube of the synthesized plasmid with 100 μL of H2O, measure the plasmid concentration using a Qubit4 fluorescence meter, and calculate the plasmid concentration according to the formula: plasmid concentration = (M × 6.02 × 1023 × 10-9) / (n × 660), where M is the plasmid DNA concentration and n is the length of the recombinant plasmid (including the vector length and the target fragment) in bp. Store the diluted positive plasmid standards at -80 °C in the refrigerator.

[0047] Methods

[0048] Singleplex digital PCR amplification

[0049] To verify whether the synthesized probe primers can be effectively amplified in the digital PCR system, conduct experiments according to the recommended 15 μL reaction system of the Pilot digital PCR universal kit: 3 μL of PCR buffer, 1.35 μL each of 10 μM upstream and downstream primers, 0.375 μL of 10 μM probe, 2 μL of DNA template (diluted positive plasmid), and make up to 15 μL with ddH2O. The reaction conditions are: pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 s, annealing at 60 or 61 °C for 1 min, for 40 cycles.

[0050] 1.4.2 Establishment and optimization of multiplex digital PCR

[0051] 1.4.2.1 Temperature verification

[0052] Perform the experiment according to the recommended 15 μL reaction system of the leading digital PCR universal kit: 3 μL of PCR buffer, 1.35 μL of each 10 μM primer, 0.375 μL of 10 μM probe, 2 μL of DNA template (diluted positive plasmid), and make up to 15 μL with ddH2O. Set 4 temperature gradients of 59 °C, 60 °C, 61 °C, and 62 °C for annealing to determine the optimal reaction temperature. The reaction conditions are pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 s, annealing at 59 - 62 °C for 1 min, for 40 cycles.

[0053] 1.4.2.2 Determination of primer and probe concentrations

[0054] Fix the recommended optimal probe concentration of the kit at 250 nmol / L, and set the primer concentrations to 200, 300, 400, 500, 600, 700, 800, and 900 nmol / L respectively. Determine the optimal primer concentration according to the distribution of combined positive and negative droplets and the detected copy number. After determining the optimal primer concentration, further set the probe concentrations to 150, 200, 250, 300, and 350 nmol / L to screen the optimal probe concentration, and finally determine the primer and probe concentrations of the primer multiplex digital PCR reaction system. Pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, for 40 cycles.

[0055] 1.4.2.3 Sensitivity experiment and reproducibility experiment

[0056] To verify the establishment of triple ddPCR for detecting the sensitivity of SFTSV, SFGR, and Ot, take 10 μL of each of the dissolved positive standards pUC57_SFTSV, pUC57_SFGR, and pUC57_Ot, add 70 μL of H2O and mix well, then perform 10-fold serial dilutions, and select 10 -6 ,10 -7 ,10 -8 ,10 -9 ,10 -10 ,10 -11 Perform multiplex ddPCR detection on different dilution gradients of plasmids, repeat each gradient 3 times, use GraphPad Prism9 to draw the standard curve, and calculate the coefficient of variation (CV) for each gradient. CV = (standard deviation ÷ mean) × 100%.

[0057] 1.4.2.4 Threshold determination

[0058] Apply the established triple ddPCR to detect 30 negative samples, 15 SFTSV positive specimens, 8 SFGR positive samples, and 2 Ot positive samples, and use MedCalc software to calculate the optimal cut-off value.

[0059] 1.4.2.5 Specificity of triple ddPCR

[0060] Using the above - determined ddPCR reaction system, detect the specificity of triple ddPCR for nucleic acid templates of hemorrhagic fever with renal syndrome virus, dengue virus, Brucella, and Streptococcus suis.

[0061] 1.5 Sample detection

[0062] Apply the established triple ddPCR to detect specimens of suspected cases of severe fever with thrombocytopenia syndrome in Chuzhou in 2024.

[0063] 2 Experimental results

[0064] 2.1 Single - plex ddPCR

[0065] In single - plex ddPCR, using the synthesized positive plasmid as the template, the three pairs of probe - primers showed good amplification effects respectively. The positive and negative droplets in each system were clearly distinguishable, and no positive amplification occurred in the negative control. All three pairs of probe - primers could effectively bind to the template, which was suitable for constructing the multiplex ddPCR system.

[0066] 2.2 Multiplex system construction

[0067] 2.2.1 Determination of reaction temperature

[0068] The ddPCR reaction is greatly affected by the annealing temperature. Too low an annealing temperature may lead to non - specific amplification, and too high an annealing temperature may reduce the reaction sensitivity. In this study, the optimal temperature of the ddPCR reaction was determined according to the separation of positive and negative droplets and the number of detected positive droplets. The results showed that the separation of positive and negative droplets was better at 60 and 61 °C. Further, we compared the detection of three positive droplets at reaction temperatures of 60 and 61 °C. The copy numbers / μL of SFTSV and Ot were higher at an annealing temperature of 60 °C, which were 124.61 and 117.77 copies / μL respectively. Therefore, 60 °C was selected as the annealing temperature of the reaction system.

[0069] 2.2.2 Determination of primer - probe concentration

[0070] According to the recommended probe concentration range of 100 - 300 nM in the kit, the probe concentration was fixed at 250 nM, and the primer concentrations were set at 200, 300, 400, 500, 600, 700, 800, 900 nM. The results showed that SFTSV and SFGR had good droplet separation at primer concentrations of 200 - 900 nM, and there was no significant difference in fluorescence values at primer concentrations of 600 - 900 nM. Ot had good separation of positive and negative droplets at 400 - 900 nM ( Figure 3)。 Further comparing the quantitative detection results, the detection value of Ot was the highest at the primer concentration of 600 nM, and the values of SFTSV and SFGR were the highest at the primer concentration of 600 nM (concentration range: 200 - 700 nM). Therefore, 600 nM was selected as the experimental primer concentration. Fixing the primer concentration at 600 nM, the probe concentrations were set at 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM respectively. The results showed that SFTSV, SFGR, and Ot had good resolution at probe concentrations between 100 - 300 nM ( Figure 3 ). We further compared the concentrations of SFTSV, SFGR, and Ot at different probe concentrations. The results showed that the detection values of the three probes were the highest at the concentration of 250 nM. Therefore, the primer concentration of 600 nM and the probe concentration of 250 nM were determined as the triple ddPCR reaction conditions.

[0071] 2.3 Sensitivity and reproducibility experiments

[0072] Mix the three positive plasmid standards and perform serial 10-fold dilutions. Detect the samples after 10 -6 ~10 -11 six gradient dilutions, with 3 replicates for each gradient, to determine the sensitivity and reproducibility of the reaction system. As shown in Table 2, the lower limits of quantification for triple digital PCR were 0.59 copies / μL for SFTSV, 0.46 copies / μL for SFGR, and 0.36 copies / μL for Ot. Using the logarithm of the theoretical copy number / μL from 10 -6 ~10 -11 as the abscissa and the logarithm of the corresponding ddPCR-detected copy number / μL as the ordinate, a standard curve was plotted. Figures 4-6 is the fitting curve graph of the linear relationship for triple ddPCR sensitivity detection; Figure 4 is the sensitivity standard curve graph of SFTSV; Figure 5 is the sensitivity standard curve graph of SFGR; Figure 6 is the sensitivity standard curve graph of Ot. The results are shown in Figure - 6. The standard curve of SFTSV is y = 1.060X - 0.4012, R 2 = 0.9800; the standard curve of SFGR is y = 1.053X - 0.3900,

[0073] R 2 = 0.9921; the standard curve of OT is y = 1.057X - 0.4490, R 2 = 0.9833. Each sample was repeated 3 times, and the coefficient of variation was small. The established triple ddPCR had high sensitivity, good reproducibility, and stable detection results.

[0074] Table 2 Detection results of sensitivity and reproducibility experiments for triple digital PCR

[0075]

[0076]

[0077] 2.4 Detection threshold determination

[0078] Serum and whole blood specimens from healthy individuals were extracted, and triple ddPCR was used to detect 30 negative samples, 15 SFTSV-positive specimens, 6 SFGR-positive samples, and 2 Ot-positive samples. The optimal cut-off value of the samples was determined by the ROC curve. The results showed that the detection threshold of SFTSV was 0.095 copies / μL, the threshold of SFGR was 0.286 copies / μL, and the threshold of Ot was 0.319 copies / μL.

[0079] 2.5 Specificity verification of triple ddPCR

[0080] Triple ddPCR was used to detect the nucleic acids of hantavirus, dengue virus, Brucella, and Streptococcus suis. The results showed that the detection results were all lower than the detection threshold. It indicates that the established triple ddPCR has good specificity and does not cross-react with other pathogens.

[0081] 2.6 Detection of clinical samples

[0082] The above-mentioned triple digital PCR and real-time fluorescence quantitative PCR were used to detect 24 clinical samples in Chuzhou City. The detection results were consistent. Among them, 12 were SFTSV-positive, and the positive rate was 50%.

[0083] The above are only the preferred embodiments for explaining the present invention, and are not intended to limit the present invention in any form. Therefore, any modification or change of the present invention made under the same inventive spirit should still be included in the scope intended to be protected by the present invention.

Claims

1. A kit for triple droplet digital PCR detection of SFTSV, SFGR and Ot, characterized in that: Includes the following primers and probes: SFTSV-L4919-38-F: 5'-CATACACWGAGGAGTACAAG-3'; SFTSV-L5004-24R: 5'-CAYARRGTCATGGTCCTWGAT-3'; Probe SFTSV-L4982P: ROX-5′-TCCTRATGACCTCTGATGGYT-3′; 17Kd-F: 5'-GGTTCTCAATTCGGTAAG-3'; 17Kd-R: 5'-TATCCTGCTCATCCATAC-3'; Probe 17Kd-P: FAM-5′-TCCACCAAGAACTGCTCCAAG-3′; htrA-670F: 5'-GTATCTTACTCAGGCATAA-3'; htrA759R: 5'-AGCATAACATTTAACATACCA-3'; Probe htrA699P: VIC-5′-TCCATCTAATACTGTACTTGAAGCA-3′; Plasmids pUC57_SFTSV, pUC57_SFGR and pUC57_Ot.

2. A triple droplet digital PCR detection method for SFTSV, SFGR and Ot, characterized by: The following steps are involved: Step 1: Singleplex digital PCR amplification PCR buffer 3 μL, 1.35 μL of 10 μM upstream and downstream primers, 0.375 μL of 10 μM probe, 2 μL of plasmid as DNA template, ddH2O to 15 μL; reaction conditions: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 60 or 61℃ annealing for 1 min, 40 cycles; Step 2: Multiplex digital PCR setup and optimization (1) Temperature verification: PCR buffer 3 μL, 1.35 μL of each 10 μM primer, 0.375 μL of 10 μM probe, 2 μL of DNA template, ddH2O to 15 μL; annealing temperature set to 59-62°C; reaction conditions: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, annealing at 59-62°C for 1 min, 40 cycles; (2) Sensitivity test and repeatability test To verify the sensitivity of triple ddPCR for detecting SFTSV, SFGR and Ot, 10 μL of dissolved positive standards pUC57_SFTSV, pUC57_SFGR and pUC57_Ot were taken, 70 μL of H2O was added and mixed, and then 10-fold dilution was performed. -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 Plasmids with different dilutions were subjected to multiplex ddPCR detection, and each gradient was repeated three times. GraphPad Prism9 was used to draw the standard curve and calculate the coefficient of variation of each gradient; (3) Threshold determination A triple ddPCR assay was established to detect 30 negative samples, 15 SFTSV-positive samples, 8 SFGR-positive samples, and 2 Ot-positive samples, and the optimal cutoff value was calculated using MedCalc software; (4) Triple ddPCR specificity The above-determined ddPCR reaction system was used to detect the triple ddPCR specificity of the nucleic acid templates of hemorrhagic fever virus, dengue virus, Brucella, and Streptococcus suis.

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