39 results about "Mycoplasma synoviae" patented technology

The invention relates to a traditional Chinese medicine preparation for preventing and treating poultry mycoplasma synoviae and a preparation method of the traditional Chinese medicine preparation. The traditional Chinese medicine preparation is prepared from the following raw materials in parts by weight: 20-30 parts of dried ginger, 10-20 parts of radix sophorae flavescentis, 20-30 parts of cassia twig, 20-30 parts of acanthopanax, 20-30 parts of eucommia ulmoides, 20-30 parts of salvia miltiorrhiza and 20-30 parts of motherwort. Seven traditional Chinese medicines are compounded and have a synergistic effect, the poultry mycoplasma synoviae can be effectively prevented and treated, the normal growth and production performance of poultry can be maintained, the safety of animal products caused by using antibacterial drugs and antibiotics for treatment can be avoided, and the traditional Chinese medicine composition is suitable for popularization and application of large-scale farms.

The invention belongs to the technical field of biological medicine, and particularly relates to a mycoplasma synoviae culture medium capable of improving strain titer and a preparation method of the mycoplasma synoviae culture medium. The mycoplasma synoviae culture medium comprises a basic culture medium and auxiliary components, the basic culture medium is prepared from HEPES, milk protein hydrolysate, MEM, beef heart extract powder, coenzyme I, 1% phenol red, magnesium sulfate heptahydrate, L-cysteine hydrochloride, lactose-N-tetrasaccharide, disodium clodronate and water; the auxiliary components comprise porcine serum and penicillin. According to the mycoplasma synoviae culture medium obtained by the preparation method, the passage time of the mycoplasma synoviae is shortened, and the strain titer is improved. The inactivated vaccine prepared from the chicken mycoplasma synoviae cultured by the culture medium is good in safety and immunogenicity, can effectively prevent infection of the chicken mycoplasma synoviae, and has a good application prospect.

The application discloses a primer probe combination for detecting chicken synovial mycoplasma based on RPA-CRISPR / Cas12 and application thereof, and belongs to the technical field of gene detection. The primer probe combination is composed of an upstream primer sequence shown as SEQ ID NO. 3, a downstream primer sequence shown as SEQ ID NO. 4 and a crRNA probe sequence shown as SEQ ID NO. 5. The primer probe combination designed in the application amplifies nucleic acids by using RPA amplification technology, and under the guidance of the crRNA probe, the product is cut by Cas12 enzyme to single-stranded DNA report sequence, and detection is carried out through a fluorescence signal, so that the chicken synovial mycoplasma can be rapidly detected in a specific manner, and the minimum detection limit can reach 1 copy / μL. The application not only can rapidly detect the result, but also solves the defects of current detection methods, such as low sensitivity, long time, high cost and the like.

The invention discloses a method and a kit for simultaneously detecting avian leukosis virus, mycoplasma synoviae and salmonella pullorum antibodies, and belongs to the technical field of animal epidemic disease detection. According to the method, an avian leukosis virus p27 protein, a chicken mycoplasma synoviae p50 protein and a truncated GroEL-delta8-1 protein of a dominant antigen GroEL of salmonella pullorum are taken as coating antigens, synchronous detection of the three antibodies is realized by optimizing ELSA reaction conditions, and a kit capable of simultaneously detecting the three antibodies is developed at the same time. The kit has the characteristics of strong specificity, high sensitivity and simplicity and convenience in operation, is suitable for rapid screening and purification of poultry epidemic diseases, and reduces the detection cost.

The invention relates to the technical field of veterinary biology, in particular to a monoclonal cell strain related to mycoplasma synoviae, a test strip and a preparation method and application of the monoclonal cell strain and the test strip. A heavy chain VH nucleotide sequence of a chicken mycoplasma synoviae antibody secreted by the monoclonal cell strain MS1C5-F8 is SEQ ID NO.1, an amino acid sequence of the chicken mycoplasma synoviae antibody is SEQ ID NO.3, a light chain VL nucleotide sequence of the chicken mycoplasma synoviae antibody is SEQ ID NO.2, and an amino acid sequence of the chicken mycoplasma synoviae antibody is SEQ ID NO.4. The chicken mycoplasma synoviae antibody not only can be subjected to specific reaction with different MS clinical isolate antigens, but also has no cross reaction with other common avian bacterial pathogens. The monoclonal antibody cell strain is used for immunizing a mouse to prepare ascites and purifying a corresponding antibody, and the purified monoclonal antibody is used for successfully preparing a colloidal gold test strip capable of detecting the MS antigen.

The invention provides a construction method and application of a chicken fallopian tube embolism model, and belongs to the technical field of animal infection models. The invention provides a construction method of a chicken fallopian tube embolism model based on multi-pathogen infection. The mycoplasma synoviae, the infectious bronchitis virus and the riemerella anatipestifer are utilized to infect the chicken, and the clinical typical salpingemphasis pathological process including cheese-like substance accumulation in a salpingemphasis cavity, serious abdominal air sacculitis complications and related tissue inflammation changes can be accurately reproduced. The model constructed by the invention is high in morbidity, and is suitable for epidemic disease pathogenesis research, drug screening and vaccine potency test. Compared with an existing single pathogen infection model, the model is closer to a clinical reality multi-pathogen cooperative infection scene, and the blank of a standardized multi-pathogen mixed infection induced fallopian tube embolism animal model is filled up.

The invention belongs to the technical field of microbial agents. The invention provides bacillus mojavensis NK-3, a microbial agent and application of the bacillus mojavensis NK-3 in cultivation. The bacillus mojavensis NK-3 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.25408, and the preservation date is June 10, 2022. The invention further provides a bacillus mojavensis microbial inoculum, and the active ingredient of the bacillus mojavensis microbial inoculum is bacillus mojavensis NK-3. The bacillus mojavensis microbial agent disclosed by the invention is applied to culture and can inhibit staphylococcus aureus; the DNA / RNA content of pathogens is reduced, and the CT value of the mycoplasma synoviae of the laying hens is increased.

The present invention provides Mycoplasma synoviae strain K5885 as deposited at the ATCC under Patent Designation PTA-127167, and progeny and derivatives thereof, for use as a vaccine for the prevention of virulent Mycoplasma synoviae infections in the birds of the order Galliformes. Also provided are compositions and methods for administration to birds of the order Galliformes.

The invention belongs to the technical field of research and development of quintuplet vaccines, and particularly relates to a chicken Newcastle disease virus strain LaSota, a chicken Newcastle disease virus strain H120, a chicken Newcastle disease virus strain N49, a chicken Newcastle disease virus strain FADV-4 and a chicken Newcastle disease virus strain HN01. The chicken Newcastle disease virus strain HN01 and the chicken Newcastle disease virus strain HN01 and the chicken Newcastle disease virus strain HN01 and the chicken Newcastle disease virus strain FADV-4 and the chicken Newcastle disease virus strain HN01 and the chicken Newcastle disease virus strain FADV-4 and the chicken Newcastle disease virus strain HN01 are combined. The chicken Newcastle disease virus LaSota strain is proliferated in chicken embryo fibroblasts or chicken kidney cells, and the chicken Newcastle disease virus LaSota strain and the preparation method thereof have the beneficial effects that the chicken Newcastle disease virus LaSota strain and the preparation method thereof can be used for preparing the chicken Newcastle disease virus LaSota strain. The preparation method comprises the following steps: selecting an inactivated newcastle disease virus LaSota strain, an inactivated bronchitis H120 strain, an inactivated bursal disease N49 strain, an inactivated fowl adenovirus FADV-4 strain and an inactivated mycoplasma synovium HN01 strain; it can be guaranteed that a follow-up vaccine has the advantages of being low in toxicity, high in biological safety, high in cross protection capacity, high in immune response speed, long in antibody protection period, high in cellular immune level and high in virus culture content.

The invention belongs to the technical field of biological agents, and discloses bacillus subtilis KY2098, a compound preparation of the bacillus subtilis KY2098 and a grape seed extract and application of the bacillus subtilis KY2098 to prevention and treatment of chicken mycoplasma synoviae. The preservation number of the bacillus subtilis is CCTCC (China Center for Type Culture Collection) NO: M 2026185. Tests prove that the bacillus subtilis and the grape seed extract compound preparation have potential interaction and can be matched with each other to play a synergistic role, the chicken mycoplasma synoviae can be effectively prevented and treated, meanwhile, the effects of enhancing the immunity of the organism and promoting growth are achieved, the treatment mode is scientific, efficient, green and environmentally friendly, and the application prospect is wide. And the method has important significance on the industrial development of antibiotic-free breeding.

The application belongs to the technical field of biological detection, and particularly relates to a SNP site for distinguishing between MS-H vaccine strains and wild strains of synovial bursa mycoplasma, a kit and application. ktrb The application first designs specific primers and MGB probes for specific conservative gene sequences, and establishes a double qPCR kit for distinguishing between MS-H vaccine strains and wild strains. Experiments in vitro prove that the kit is simple to operate, high in sensitivity, strong in specificity, and good in repeatability. The gene site and the detection kit have the advantages of avoiding the deficiency that the commonly used genes are prone to revert mutation, can be used for clinical rapid diagnosis of MS vaccine strains and wild strains, improve the accuracy of detection, promote population purification, and have certain commercial value.

This invention discloses a triple inactivated vaccine containing Mycoplasma gallisepticum, Mycoplasma synoviae, and Infectious Laryngotracheitis in chickens, its preparation method, and its application. The technical solution includes inactivated Mycoplasma gallisepticum antigen, inactivated Mycoplasma synoviae antigen, and inactivated Infectious Laryngotracheitis Virus gD protein; wherein the inactivated Mycoplasma gallisepticum antigen is prepared from Mycoplasma gallisepticum strain YBMG1, and the preservation number of Mycoplasma gallisepticum strain YBMG1 is CCTCC M 20261107. This invention rationally combines Mycoplasma gallisepticum antigen, Mycoplasma synoviae antigen, and chicken infectious laryngotracheitis virus gD protein, and uses saponin as an immune enhancer. This effectively reduces the interference of chicken infectious laryngotracheitis virus gD protein on the immune response of Mycoplasma gallisepticum and Mycoplasma synoviae, and the effect is significantly better than other polysaccharide or polypeptide immune enhancers, thereby achieving simultaneous prevention and control of Mycoplasma gallisepticum disease, Mycoplasma synoviae disease, and chicken infectious laryngotracheitis.

This invention provides a composite adjuvant, its preparation method, and its application in vaccine preparation, belonging to the field of vaccine adjuvant technology. The composite adjuvant of this invention has low viscosity, only 12 mPa·s at 20°C, ensuring smooth injection; it exhibits high biocompatibility, with no deaths or organ abnormalities in acute toxicity experiments in mice, and no local or systemic adverse reactions or stress responses in chickens after injection; it also demonstrates excellent immunogenicity, achieving a 90% seroconversion rate for Mycoplasma cambogia and an 80% seroconversion rate for Mycoplasma synoviae 28 days after immunization with an inactivated Mycoplasma synoviae vaccine.

The invention discloses a fluorescence immunochromatography test strip for quantitatively detecting a chicken mycoplasma synoviae antibody, the fluorescence immunochromatography test strip comprises a bottom plate, a sample pad, a fluorescent pad, a nitrocellulose membrane and an absorption pad are sequentially arranged on the bottom plate, the fluorescent pad is a time-resolved fluorescent microsphere labeled chicken mycoplasma synoviae MSPB protein, and the nitrocellulose membrane is a nitrocellulose membrane. A detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is chicken mycoplasma synoviae MSPB protein, and the quality control line is goat anti-chicken IgG, the MSPB protein is successfully prepared, can be specifically combined with a chicken mycoplasma synoviae antibody, and does not react with other poultry virus antibodies.

The invention discloses a recombinant chicken mycoplasma synoviae antigen protein as well as a coding gene, a preparation method and application thereof. A prokaryotic expression system is used for expressing and purifying rRS02775 recombinant protein, rRS01455 recombinant protein, rRS01745 recombinant protein, rRS01040 recombinant protein, rRS01750 recombinant protein and rRS02145 recombinant protein respectively, further, the multi-antibody serum prepared by immunizing BALB / c mice has a remarkable growth inhibition effect on a chicken mycoplasma synoviae WVU1853 strain, a Ningxia / 2017-1 strain and a Shandong / 2017-2 strain, the highest valence of a neutralizing antibody can reach 1: 256, and the multi-antibody serum has the potential of being used for developing chicken mycoplasma synoviae subunit vaccines.

The invention belongs to the technical field of antibody detection kits, and relates to an application of chicken mycoplasma synoviae recombinant protein FBA in preparation of a chicken mycoplasma synoviae antibody indirect ELISA detection kit, which is characterized in that the amino acid sequence of the chicken mycoplasma synoviae recombinant protein FBA is as shown in SEQ ID NO.1. The indirect ELISA detection kit for the chicken mycoplasma synoviae antibody is characterized by comprising an elisa plate which takes chicken mycoplasma synoviae recombinant protein FBA as a coating antigen, wherein the amino acid sequence of the chicken mycoplasma synoviae recombinant protein FBA is shown as SEQ ID NO.1. The method is helpful for deeply developing the research on chicken mycoplasma synoviae infection and immunity, and a rapid, sensitive and good-specificity method is provided for clinical diagnosis of chicken mycoplasma synoviae infection.

The invention discloses a traditional Chinese medicine composition as well as a preparation method and application thereof in preventing and treating mycoplasma synoviae, and belongs to the technical field of traditional Chinese veterinary medicines. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 20 to 40 parts of cortex phellodendri, 10 to 20 parts of rhizoma smilacis glabrae, 10 to 20 parts of liquorice root, 2 to 5 parts of rhizoma anemarrhenae, 3 to 8 parts of radix angelicae sinensis and 5 to 10 parts of rhizoma alismatis. In addition, the invention also provides a preparation method of the traditional Chinese medicine composition, which comprises the following steps: mixing the raw materials, adding a first solvent, soaking to obtain a mixture, heating the mixture at 50-60 DEG C, heating the mixture at 90-100 DEG C, filtering, and concentrating under reduced pressure to obtain the traditional Chinese medicine composition. In addition, the invention also provides application of the traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the preparation method in preventing and treating mycoplasma synoviae. According to the invention, the mycoplasma synoviae is safely and effectively prevented and treated.

The present invention discloses a recombinant Mycoplasma synoviae antigen protein, its coding gene, preparation method and application. The present invention respectively expresses and purifies rRS02775, rRS01455, rRS01745, rRS01040, rRS01750, rRS02145 recombinant proteins by using a prokaryotic expression system. Furthermore, the polyclonal antiserum prepared by immunizing BALB / c mice has a significant growth inhibitory effect on Mycoplasma synoviae strains WVU1853, Ningxia / 2017-1, and Shandong / 2017-2. The neutralizing antibody titer can reach up to 1:256 at most, and it has the potential to be developed into a Mycoplasma synoviae subunit vaccine.

The invention relates to the technical field of microbial fermentation, and particularly provides a liquid culture medium and a fermentation method of mycoplasma synoviae. The liquid culture medium is composed of a basic culture medium containing 1-20 g / L of pplo broth and 0.2-3 g / L of poloxamer. According to the application, the poloxamer is added into the culture medium of the mycoplasma synoviae for the first time, so that the culture density of the mycoplasma synoviae is improved, and the poloxamer is accidentally found to improve the immunogenicity of MS (Murashige and Skoog). In addition, when the liquid culture medium is used for fermenting the mycoplasma synoviae, the fermentation time is shortened to 12-14 hours.

The application provides a mycoplasma gallisepticum triple subunit vaccine and application thereof, and the vaccine comprises EF-TU protein with an amino acid sequence as shown in SEQ ID NO. 1, PdHD protein with an amino acid sequence as shown in SEQ ID NO. 2, MS1360 protein with an amino acid sequence as shown in SEQ ID NO. 3 and a pharmaceutically acceptable adjuvant. The application realizes antigen epitope complementation and immune synergistic enhancement by screening the optimal vaccine formula through two-by-two mixed combination and three-protein concentration gradient mixed system screening of EF-TU, PdHD and MS1360 three high immunogenic membrane proteins, and solves the problems of low protection rate and unstable immune effect of the existing vaccine.

The present invention provides a Mycoplasma synoviae of chicken, a resulting live vaccine, a preparation method thereof and an application, belonging to the technical field of veterinary vaccines. The Mycoplasma synoviae of chicken is the Mycoplasma synoviae MS 7 strain, which was deposited at the China Center for Type Culture Collection on July 10, 2024, and its deposit number is CCTCC M 20241533. Preparing a live vaccine from the Mycoplasma synoviae MS 7 strain of chicken has a lower cost, is more suitable for industrial production, is convenient for storage and transportation, has a simple usage method, and is conducive to large-scale popularization and use. Compared with the attenuated vaccine prepared by fully emulsifying the existing MSHLB-200 strain and white oil for injection and the commercial vaccine MS-H, it is convenient for storage and transportation and has an ideal immune effect.

The invention provides a chicken mycoplasma synoviae subunit vaccine, a preparation method, detection and application thereof. The chicken mycoplasma synoviae VY930 protein expressed on the basis of an escherichia coli prokaryotic expression system has good immunogenicity, and a subunit vaccine containing the protein is prepared from the chicken mycoplasma synoviae VY930 protein so as to effectively prevent and control chicken mycoplasma synoviae infection. Animal challenge protection tests prove that the subunit vaccine has immune protection potential equivalent to or slightly superior to that of an inactivated vaccine, and clinical symptoms caused by the mycoplasma synoviae can be remarkably relieved. The subunit vaccine disclosed by the invention is simple and convenient in preparation process and low in cost, can adapt to a large-scale production mode, and has the potential of preventing and controlling clinical infection of the mycoplasma synoviae and promoting a population purification process. The invention further provides a monoclonal antibody aiming at the protein, the monoclonal antibody can be used as an index for judging whether immunization is carried out or not, meanwhile, the immune protection evaluation criterion after chicken mycoplasma synoviae foot pad challenge is refined, and a meticulous measurement standard is provided for follow-up vaccine research and development.

The application belongs to the technical field of microorganism detection, and particularly relates to a primer probe combination, a kit and application for detecting chicken synovial capsule mycoplasma based on RAA-LFD. The nucleotide sequence of the upstream primer in the primer probe combination is shown in SEQ ID NO. 3, the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 4, and the nucleotide sequence of the probe is shown in SEQ ID NO. 10. Based on the primer probe combination, the application further develops a kit and a method for detecting chicken synovial capsule mycoplasma. The method has high detection accuracy, good specificity and high sensitivity, can rapidly and accurately realize on-site rapid visual detection of chicken synovial capsule mycoplasma, and is particularly suitable for use in the field.

The application provides a Mycoplasma synoviae subunit vaccine, a preparation method, detection and application thereof. The Mycoplasma synoviae VY930 protein expressed based on an Escherichia coli prokaryotic expression system has good immunogenicity, and a subunit vaccine containing the protein is prepared, so as to effectively prevent and control Mycoplasma synoviae infection. It is proved by animal challenge protection test that the subunit vaccine has equivalent or slightly better immunoprotective potential than the inactivated vaccine, and can significantly reduce the clinical symptoms caused by Mycoplasma synoviae. The subunit vaccine has the potential of preventing and controlling Mycoplasma synoviae clinical infection and promoting the purification process of the population. The application also provides a monoclonal antibody for the above protein, which can be used as an index for judging whether immunization is performed or not, and further refines the immune protection evaluation criteria after footpad challenge of Mycoplasma synoviae, so as to provide a detailed measurement standard for subsequent vaccine research and development.

The invention discloses a traditional Chinese medicine composition and application, and relates to the technical field of traditional Chinese medicine, the traditional Chinese medicine composition comprises, by weight, 1 part of sinomenine and 0.5-1.5 parts of formononetin, and the traditional Chinese medicine composition is used for treating chicken mycoplasma synoviae. As an innovative therapy for treating chicken mycoplasma synoviae, the traditional Chinese medicine composition provides an efficient and resistance-free treatment choice for farmers due to the characteristics of simple formula and easiness in obtaining. The traditional Chinese medicine composition is only composed of two traditional Chinese medicines, the cost is reduced, the preparation process is simplified, and large-scale application becomes possible. More importantly, the composition avoids negative effects caused by use of antibiotics and is especially suitable for the situation that antibiotics cannot be abused in laying hen breeding, a breeder can effectively control diseases and reduce economic losses under the situation that food safety regulations are not violated, the safety of poultry products is guaranteed, and the requirements of consumers for antibiotic-free products are met.

The invention belongs to the field of microbial culture media. The invention provides a mycoplasma synoviae culture medium and a preparation method thereof, the culture medium comprises oat hydrolysate, brain heart extract powder, yeast extract powder, beef extract powder and specific nutritional factors, and the specific nutritional factors comprise horse serum, vitamins, arginine and glucose. The invention also provides a preparation method of the culture medium. The culture medium contains basic nutrient substances and specific nutritional factors required by growth of the mycoplasma synoviae, under the synergistic effect of the components, the viable bacteria titer is remarkably improved, high titer is achieved, the serum dosage is remarkably small, the influence of exogenous factors in serum on mycoplasma products is reduced, and the culture medium is more suitable for production.

The invention belongs to the technical field of feed additives, and provides a functional additive Qiwei egg-strengthening powder and application thereof. The functional additive Qiwei Zhengegg powder comprises astragalus membranaceus, schisandra chinensis, glossy privet fruit, leonurus, rehmannia, gardenia and liquorice. The functional additive Qiwei Zhengshan obviously improves the feed utilization rate and the laying rate, effectively improves the color and glossiness of eggshells, improves the quality of the eggshells in the laying period, reduces sun eggs, sand-preserved eggs, soft eggs, shell-broken eggs and the like caused by reproductive mycoplasma synovialis, improves the egg yield, prolongs the egg laying peak period, reduces the feed-egg ratio and improves the egg quality. And good economic benefits are achieved.

The invention relates to the field of poultry vaccine preparation, in particular to a new tributary glandular smooth quintuplet inactivated vaccine and a preparation method thereof.The vaccine is prepared from, by mass, 2-3 parts of a water phase and 3-5 parts of an oil phase; wherein the water phase is prepared from the following components in parts by mass: 10 to 25 parts of adjuvant; 10-20 parts of a first antigen containing inactivated avian influenza virus; 10-20 parts of a second antigen containing inactivated infectious bronchitis virus; 10-20 parts of a third antigen comprising an inactivated Newcastle disease virus; 10 to 20 parts of a fourth antigen containing inactivated chicken mycoplasma synoviae; the composition comprises 10-20 parts of a fifth antigen of inactivated fowl adenovirus, and the total parts are 100. The vaccine prepared by the invention does not generate any adverse reaction to chickens, is high in experimental safety, and can induce high-level neutralizing antibodies in immunized chickens, which indicates that the immunized chickens can effectively resist attack of Newcastle disease virus, avian influenza, infectious bronchitis virus, adenovirus and mycoplasma synoviae.

The invention relates to the technical field of reagent detection, and discloses a chicken mycoplasma synoviae plate agglutination detection system as well as a detection method and application thereof. The detection sensitivity is improved, and the revolutionary breakthrough of the detection sensitivity is realized by constructing a'biotinylated recombinant antigen-streptavidin-color microsphere 'cascade signal amplification system; the detection specificity is high, cross misjudgment is effectively avoided, the high-purity and high-specificity genetic engineering recombinant p80 / vlhA fusion protein is adopted as a core recognition element, and interference of non-target impure protein is completely eradicated from an antigen source; the result interpretation is visual and objective, and the detection result is presented in the form of red particles or flocculent agglutination which can be clearly seen by naked eyes, and is clearly compared with a uniform red opacifying background.

This invention relates to the field of identification technology for Mycoplasma synoviae MS-H strain and wild-type strain, specifically a LAMP primer-probe composition and kit for identifying Mycoplasma synoviae MS-H strain and wild-type strain. It includes an upstream outer primer with the nucleotide sequence shown in SEQ NO:1, a downstream outer primer with the nucleotide sequence shown in SEQ NO:2, an upstream inner primer with the nucleotide sequence shown in SEQ NO:3, a downstream inner primer with the nucleotide sequence shown in SEQ NO:4, an MS-H strain probe with the nucleotide sequence shown in SEQ NO:5, and an MS wild-type strain probe with the nucleotide sequence shown in SEQ NO:6. This invention uses MGB to enhance the thermostability of the probe and combines RNase H2-mediated blocking probe specific activation technology with an enhancer to improve the specificity of the reaction system, enabling LAMP to recognize single-base differences at isothermal conditions, with a detection limit reaching 1 copy / µL.