CN101979665A - Multiple polymerase chain reaction (PCR) kit and method for detecting mosquito-borne pathogens - Google Patents
Multiple polymerase chain reaction (PCR) kit and method for detecting mosquito-borne pathogens Download PDFInfo
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Abstract
The invention provides a multiple polymerase chain reaction (PCR) kit for detecting mosquito-borne pathogens. The kit comprises six pairs of specific primers. The invention also provides a method for detecting the mosquito-borne pathogens. Electrophoresis is performed on a PCR-amplified product. Whether pathogens, such as encephalitis B virus, dengue fever virus, yellow fever virus, plasmodium falciparum, plasmodium vivax, plasmodium knowlesi, plasmodium ovale, plasmodium malariae, wuchereria malayi, wuchereria bancrofti and the like, exists or not is detected and identified according to the length of a PCR-amplified fragment. By the method, various reported mosquito-borne pathogens, and the yellow fever virus and west nile virus which come from other countries can be detected quickly, accurately and sensitively at the same time, and can be applied to the detection of various samples, such as mosquitoes, blood of patients, tissue fluid and the like. The invention provides a low-cost and high-efficient method for early monitoring and finding mosquito-borne disease prevalence for prevention and control work of mosquito-borne diseases in China.
Description
Technical field:
The present invention relates to field of biological detection, relate in particular to the gene test of mosquito matchmaker pathogenic agent (encephalitis b virus, dengue fever virus, plasmodium, filaria), particularly a kind of multiple PCR reagent kit and detection method that detects mosquito biography pathogenic agent.
Background technology:
In China popular or once popular mosquito matchmaker disease malaria, filaricide, encephalitis B, singapore hemorrhagic fever etc. are arranged.It is one of effective measure of popular, the pre-mosquito-proof matchmaker's disease of early discovery that detection mosquito matchmaker carries pathogenic agent.
Mosquito is the communication media of multiple transmissible diseases such as malaria, filaricide, encephalitis B, singapore hemorrhagic fever.Mostly the monitoring of mosquito matchmaker disease, research in the past are should take all factors into consideration for the control of mosquito matchmaker disease at single pathogen detection of planting.Come monitor pathogen from mosquito matchmaker aspect, the method that belongs to early monitoring and discovery transmissible disease, the half-nest type multiplex PCR detects mosquito matchmaker pathogenic agent method will provide a kind of monitoring method of early stage comprehensive high-efficiency for the sick control of mosquito matchmaker, and the effective early warning and the control of mosquito matchmaker disease is had bigger value.
At present, still have more than 100 country to be the popular district of malaria in the world, about 2,200,000,000 people are subjected to the threat of malaria, and 300～5,000,000 malaria clinical cases are arranged every year, and death toll is 110～2,700,000.Malaria also is serious harm China people's health, influences the great parasitosis of socio-economic development.Through insistent positive control for many years, China is popular in the control malaria, reduce and to have obtained remarkable effect aspect the hazard rating.Because China's all kinds of epidemic factors in malaria epidemic-stricken area not radical change as yet, the floating population increases severely and causes contagium diffusion and accumulation, climate warming, variation of ecology and environment cause media to increase by mosquito density, generation and the diffusion of plasmodium falciparum and media anopheles to preventing and treating drug resistance, these problems have had a strong impact on the process of China's malaria control work, and the malaria morbidity number is unsteady state in recent years.Health ministry has been formulated the plan of eliminating malaria, for guaranteeing the enforcement of this plan, should planned, continuously, systematically carry out malaria epidemic situation, monitoring, grasp malaria regularty of epidemic and trend, provide scientific basis for estimating prevention effect and working out the malaria control countermeasure.Effectively mosquito matchmaker detection is the important technology guarantee of strengthening China's prevention and control malaria, is the important content of effectively carrying out prevention and the work of control malaria.
Lymph filaricide is one of parasitosis of serious harm people ' s health, in China's popular filaricide two kinds of bancroftosis and filariasis malayis is arranged, and is caused by wuchereria bancrofti and cloth Shandong, Malaysia nematode respectively.Once popular in 15 provinces (city, district), compromised population reaches 3.3 hundred million.Through the control in 40 years, realized national basic elimination filaricide.Existing report, in economy, the relatively poor outlying poverty-stricken area of sanitary condition, and the area that has weak link in the control, monitoring, border on the area with the neighbouring country popular district of filaricide, external floating population gathering area is even only exist discrete high-density microfilaremia person just can cause that in the locality filaria newly infects.The disease of going out is engaged in to the greatest extent, prevents that filaricide is revivable in China, eliminates the filaricide area and still need carry on monitoring.
For flavivirus, be example with encephalitis, singapore hemorrhagic fever.Encephalitis is to be caused by encephalitis b virus, by the acute infectious disease of killing propagation.Encephalitis lethality rate and disability rate height are particularly one of the main transmissible diseases of children's health of threat crowd.Be onset peak season summer and autumn, popularly the district distributes closely related with the distribution of media mosquito, China once was the popular district of encephalitis height, once be very popular in the sixties in 20th century and the initial stage seventies whole nation, along with the ring vaccination Vaccinum Encephalitidis Epidemicae, the encephalitis sickness rate obviously descends after the seventies, maintain lower morbidity level in recent years, whole nation encephalitis reported cases are counted every year between 5000～10000 examples, but because the blind area or the other reasons of vaccine inoculation have outbreak of epidemic during partial area.
Dengue virus has 1～4 type, propagates through Aedes aegypti and Aedes albopictus.The Category B notifiable disease that is defined as according to " People's Republic of China's law on the prevention and control of infectious diseases ".It is a very important public health problem of the torrid zone, subtropical zone.At present, have 2,500,000,000 people to be subjected to the threat of dengue virus infection in the world approximately, annual generation dengue virus infection patient surpasses 100,000,000 people, and has 500,000 people to develop into dengue hemorrhagic fever or dengue shock syndrome, causes about 25000 people's death.China is since the nineties in 20th century, this disease mainly in Guangdong, Fujian is popular, mostly be popular on a small scale or distribute.1999 and 2004 are because of introduced cases cause Fujian and zhejiang and other places generation outbreak of epidemic, and other provinces and regions also often have the generation of introduced cases in recent years.Because that dengue virus is propagated is swift and violent, sickness rate is high, particularly in recent years because flow of personnel is frequent and international tourism's fast development, the distribution range that makes the popular scope of dengue virus and communication media Aedes aegypti and Aedes albopictus is also in corresponding expansion.Often there be the alternately popular of different serotypes virus in dengue virus in an area, and this has more increased the threat of dengue hemorrhagic fever and dengue shock syndrome generation.The case fatality rate of dengue hemorrhagic fever and dengue shock syndrome is higher, not only has a strong impact on people health, and has a strong impact on the development of local economy, trade and tourist industry.Implement the mosquito matchmaker monitoring of epidemic encephalitis type B and singapore hemorrhagic fever, help to find in advance epidemic situation, scientifically effective prophylactico-therapeutic measures is in time taked in prediction, early warning, reduces sickness rate.
Monitor plasmodium filaria flavivirus from mosquito matchmaker aspect and infect, belong to the method for early monitoring and discovery disease transmission in monitoring of infectious disease, the mosquito matchmaker detects for effective early warning of mosquito matchmaker disease and control important meaning.Research in the past all is at a kind of pathogen detection, should take all factors into consideration for the control of mosquito matchmaker disease.
Easy accurate test method is to find that mosquito passes the important technology guarantee of pathogenic agent, grasps its regularty of epidemic and trend and provides scientific basis (Nuchprayoon et al., 2003) for estimating prevention effect and working out Preventing Countermeasures.Also can be used for the clinical detection that mosquito passes disease.Monitoring mosquito infection conditions has material impact (Gage et al., 2008) to malaria and filaricide control.Diagnostic wire parasitosis and malaria, traditional method are sediments microscope inspections, though this method is convenient, expense is cheap and relatively accurate, often need to be skilled in technique and time-consuming work, and are not enough to detect plasmodium low density blood sample.For solving these deficiencies, developed immunological method, as enzyme linked immunosorbent assay (ELISA) and immunosorption chromatography (ICT), being used for accurate assessment blood sample and mosquito colony pathogenic agent carries situation [Nuchprayoon et al., 2003, Wirtz et al., 1985].Developed simpler, more responsive diagnostic method later on again, promptly based on more advanced immune immunosorption chromatography (ICT) and polymerase chain reaction (PCR) technology.As the bancroft's filaria (Weil et al., 1997) in can the rapid detection blood examination negative blood sample of extremely sensitive species specificity immunosorption chromatography (ICT); Similar techniques has been developed and has been used for plasmodium and the interior sporozoite (Bangs et al., 2002, Wirtz et al., 1985, Wongsrichanalai, 2001) of mosquito body in the human body.
Use responsive specific detection technology such as PCR can obtain disease popularity situation accurately.Plasmodium, filaria DNA can reflect crowd's infection conditions (Bockarie et al., 2000, Chanteau et al., 1994 indirectly in the detection mosquito body, Goodman et al., 2003, Ramzy et al., 1997, Rao et al., 2006, Rougemont et al., 2004, Vasuki et al., 2008, Chansiri and Phantana, 2002, Farid et al., 2001, Williams et al., 2002).
Is target gene with the RFLP-PCR method with bancroft's filaria SspI 188bp reiterated DNA sequences, can detect the bancroft's filaria genomic dna (Chansiri and Phantana, 2002, Farid et al., 2001, Williams et al., 2002) of 0.1pg.(internal transcribed spacer1, ITS-1) sequence can detect multiple filaria (Nuchprayoon et al., 2005) for target gene RFLP-PCR to transcribe 1 district, interval with filaria.Use the multiplex PCR method to detect bancroft's filaria and Wuchereria malayi (Chansiri etal., 2001, Mishra et al., 2005, Mishra et al., 2007) in mosquito and the blood sample.Nest-type PRC susceptibility height is compared it with conventional P CR and can be detected more micro-filaria, rarely has report but use nest-type PRC to detect filaria at present.Along with delivering of bancroft's filaria and Wuchereria malayi ITS-1 gene order, can on this highly repetitive sequence, design primer, set up the various new detection method of PCR-based principle.
In the plasmodium context of detection, immunity-chromatography test as the selection of diagnosis malaria, can not be used for routine diagnosis (Humar et al., 1997, Tham et al., 1999) but its sensitivity is not enough.Dna probe is compared microscopy sensitivity and is increased (Waters and McCutchan, 1989), but this method is unsuitable for the screening of great amount of samples.It is target gene (Li et al., 1995 with little ribosomal subunit DNA (SSU rDNA) that PCR detects many; Schindler et al., 2001; Snounou et al., 1993).Because plasmodium SSU rDNA gene copy number is less, each genome 4-8 copy (Goman et al., 1991), nest-type PRC is used to detect micro-protozoon (Rubio et al., 1999; Schindler et al., 2001; Snounou et al., 1993; Zalis et al., 1996).Nest-type PRC is higher than conventional P CR specificity and sensitivity, but nest-type PRC is a twice PCR, step comparatively loaded down with trivial details (Picken et al., 1996); Single tube heminested PCR (Single tube hemi-nested PCR, STHN PCR) step is simple, but susceptibility is not as good as two step nest-type PRCs (Montenegro et al., 2004).Real-time PCR is that target gene detects 4 kinds of plasmodiums in the human blood sample with 18S rDNA, highly sensitive high specificity, but real-time PCR cost is higher, is not suitable for the screening (Rougemont et al., 2004) of great amount of samples.
Though have many about detecting molecular diagnosis (Chow et al., 1993, the Fulop et al. of mosquito specific kind of matchmaker's Flavivirus or genus, 1993, Tanaka, 1993, Chang et al., 1994, Puri et al., 1994, Pierre et al., 1994, Meiyu et al., 1997, Kuno, 1998), but up to now only the genus specificity heminested PCR of Scaramozzino and Lanciotti report has than hypersensitivity.Pyke (2004) has detected australian encephalitis virus with TaqMan RT-PCR, Kong (2006) with TaqManreal-time one-step RT-PCR detection by quantitative dengue virus, Dyer (2007) uses with quadrat method and has set up the method that detects arthropod-borne Flavivirus, domestic Zhou Xiaojun etc. (2008) have set up the method for SYBR Green I Real-time PCR detection Flavivirus, this technology has realized that not only PCR is from qualitative not quantitative leap, and compare with conventional PCR, it is stronger that it has specificity, effectively solve the PCR pollution problem, characteristics such as level of automation height, but shortcoming then is that machine and reaction consumptive material expense are higher relatively, causes to reach universal at present in the use.In a word, not high for the most sensitivity of detection of mosquito biography pathogenic agent both at home and abroad, cost is higher, and can not detect the multiple pathogenic agent that mosquito is carried simultaneously.The present invention also can be used for the preliminary evaluation of west nile virus, yellow fever virus.
Summary of the invention:
The object of the present invention is to provide a kind of multiple PCR reagent kit and detection method that mosquito passes pathogenic agent that detect, described this detection mosquito passes the technical problem that the multiple PCR reagent kit of pathogenic agent and detection method will solve that the method sensitivity that detection mosquito of the prior art passes pathogenic agent is not high, cost is higher and can not detect the multiple pathogenic agent that mosquito carries simultaneously.
A kind of multiple PCR reagent kit that detects mosquito biography pathogenic agent of the present invention, contain following primer:
1) primer of detection flavivirus first round PCR
The sequence of upstream primer shown in SEQ ID NO:1,
The sequence of downstream primer is shown in SEQ ID NO:2;
2) detect the primer that flavivirus second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:3,
The sequence of downstream primer is shown in SEQ ID NO:4;
3) primer of detection plasmodium first round PCR
The sequence of upstream primer shown in SEQ ID NO:5,
The sequence of downstream primer is shown in SEQ IDNO:6;
4) detect the primer that plasmodium second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:7 or shown in SEQ ID NO:8,
The sequence of downstream primer is shown in SEQ ID NO:9;
5) primer of detection Wuchereria malayi first round PCR
The sequence of upstream primer shown in SEQ ID NO:10,
The sequence of downstream primer is shown in SEQ ID NO:11;
6) detect the primer that Wuchereria malayi second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:12,
The sequence of downstream primer is shown in SEQ ID NO:13.
Further, described flavivirus comprises encephalitis b virus or dengue fever virus 1 type or dengue fever virus 2 types or dengue fever virus 3 types or dengue fever virus 4 types, west nile virus and yellow fever virus.
Further, described plasmodium comprises plasmodium falciparum or Plasmodium vivax or Plasmodium knowlesi or Plasmodium ovale or malariae.
Further, also contain polymerase chain reaction reagent in the described test kit.
Further, also contain negative quality control standard product in the described test kit.
Further, also contain the positive criteria product in the described test kit.
Further, described negative quality control standard product are sterilization ddH
2O.
Further, described positive criteria product are encephalitis B cDNA or dengue fever virus cDNA or Plasmodium vivax DNA or plasmodium falciparum DNA.
Further, each component is in the reagent of described polymerase chain reaction:
1) 10 times of polymerase chain reaction Huan Red liquid;
2) four kinds of deoxyribonucleotides, the concentration of four kinds of deoxyribonucleotides is 25mM;
3) bovin serum albumin BSA, the concentration of bovin serum albumin BSA is 5mg/ml;
4) EXTaq enzyme, the concentration of EXTaq enzyme are 5U/ μ L;
5) sterilization distilled water.
Further, the composition of described 10 times of polymerase chain reaction Huan Red liquid is as follows:
Tris-HCl 100mM
KCl 500mM
MgCl
2 15mM。
Further, the concentration of all primers in the described test kit is 10 μ M.
The present invention also provides a kind of method that mosquito passes pathogenic agent that detects, comprise a step of extracting mosquito nucleic acid, also comprise a step that the nucleic acid that extracts is increased on the pcr amplification instrument, comprise that also a product with amplification carries out the step of electrophoresis detection, also comprise and determine that mosquito passes the order-checking step of the concrete kind of pathogenic agent.
Further, in a step that the nucleic acid that extracts is increased on the pcr amplification instrument,
1) primer of detection flavivirus first round PCR
The sequence of upstream primer shown in SEQ ID NO:1,
The sequence of downstream primer is shown in SEQ ID NO:2;
2) detect the primer that flavivirus second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:3,
The sequence of downstream primer is shown in SEQ ID NO:4;
3) primer of detection plasmodium first round PCR
The sequence of upstream primer shown in SEQ ID NO:5,
The sequence of downstream primer is shown in SEQ ID NO:6;
4) detect the primer that plasmodium second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:7 or shown in SEQ ID NO:8,
The sequence of downstream primer is shown in SEQ ID NO:9;
5) primer of detection Wuchereria malayi first round PCR
The sequence of upstream primer shown in SEQ ID NO:10,
The sequence of downstream primer is shown in SEQ ID NO:11;
6) detect the primer that Wuchereria malayi second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:12,
The sequence of downstream primer is shown in SEQ IDNO:13.
The present invention also provides the above-mentioned a kind of application of multiple PCR reagent kit in detecting encephalitis b virus or dengue fever virus 1 type or dengue fever virus 2 types or dengue fever virus 3 types or dengue fever virus 4 types or west nile virus and yellow fever virus/plasmodium falciparum or Plasmodium vivax or Plasmodium knowlesi or Plasmodium ovale or malariae/Wuchereria malayi or bancroft's filaria that detects mosquito biography pathogenic agent.
The present invention and prior art are compared, and its effect is actively with tangible.The present invention has set up a kind of fast effectively molecular assay method to above-mentioned mosquito matchmaker pathogenic agent, for the early stage effectively early warning of mosquito matchmaker disease provides reliable information material.The present invention has set up the half-nest type multiplex PCR and has detected the method that mosquito passes pathogenic agent (plasmodium, filaria, flavivirus), and the method for a kind of cheap early monitoring efficiently and discovery disease transmission will be provided for the sick preventing and controlling of China mosquito matchmaker.The half-nest type multiplex PCR detects mosquito matchmaker pathogenic agent method will provide a kind of monitoring method of early stage comprehensive high-efficiency for the sick control of China mosquito matchmaker, and the effective early warning and the control of mosquito matchmaker disease is had bigger value.
The present invention can be applied to the detection of several samples, as mosquito, patient blood, tissue juice etc.Use test kit of the present invention can be fast, sensitive, detect the multiple pathogenic agent that mosquito is carried simultaneously: comprise multiple pathogenic agent such as encephalitis b virus, dengue fever virus 1-4 type, yellow fever virus, plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale, malariae, Wuchereria malayi.Thereby, provide effective early warning data to pre-mosquito-proof matchmaker's disease for the sick preventing and controlling of China mosquito matchmaker provide a kind of cheap early monitoring efficiently and find the sick popular method of mosquito matchmaker.
Description of drawings:
Fig. 1 adopts test kit of the present invention to detect the figure as a result that mosquito passes plasmodium (plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale, malariae and Wuchereria malayi);
M:DNA molecular criteria Marker I; K: negative control (ddH
2The OPCR product); 1: Wuchereria malayi and Plasmodium yoelii dna profiling PCR product in the mosquito; 2: Hainan patient's blood sample plasmodium falciparum dna profiling PCR product; 3: Huaibei patient's filter paper bloodstain plasmodium dna profiling PCR product; 4: Huaibei patient's filter paper bloodstain plasmodium dna profiling PCR product; 5: Wuchereria malayi dna profiling PCR product; 6: Wuchereria malayi dna profiling PCR product (too much when first round PCR cycle index, as to take turns the band 431bp that can occur first round PCR in the PCR product) second.
Fig. 2 adopts test kit of the present invention to detect flavivirus (encephalitis b virus, dengue fever virus 1-4 type, yellow fever virus);
M:DNA molecular criteria Marker I; K: negative control (ddH
2The OPCR product); 1: encephalitis b virus cDNA template PCR product (too much when first round PCR cycle index, as to take turns the band 260bp that can occur first round PCR in the PCR product) second; 2: encephalitis b virus cDNA template PCR product; 3: encephalitis b virus cDNA template PCR product; 4: encephalitis b virus cDNA template PCR product; 5: DEN-1 cDNA template PCR product; 6: singapore hemorrhagic fever II C-type virus C cDNA template PCR product; 7: singapore hemorrhagic fever III C-type virus C cDNA template PCR product; 8: singapore hemorrhagic fever IV C-type virus C cDNA template PCR product.
Embodiment:
Further the present invention will be described for following embodiment.
The sample that adopts among the following embodiment, animal and reagent are as follows:
The mosquito sample that infects Wuchereria malayi is from parasite institute of Chinese Disease Control and Prevention Center, mosquito flavivirus nutrient solution is from prevailing disease teaching and research room of The 2nd Army Medical College, protista teaching and research room of anopheles stephensi The 2nd Army Medical College raises, the plasmodium that the plasmodium falciparum sample picks up from Yunnan and Hainan for this teaching and research room is cultivated product, the Plasmodium vivax sample picks up from Huaibei vivax malaria patient filter paper blood sample for this teaching and research room, Plasmodium yoelii is the Plasmodium yoelii sample that protista teaching and research room of The 2nd Army Medical College liquid nitrogen is preserved, the Kunming small white mouse that mouse is provided by the The 2nd Army Medical College Experimental Animal Center.
Molecular biology reagent: tissue DNA extracts test kit, dna molecular standard Marker I (sky, Beijing root), deoxynucleoside triphosphate (dNTP), ExTaq archaeal dna polymerase, Loading buffer (TaKaRa), the Quant cDNA first chain synthetic agent box (QIAGEN), agarose (Spain), little ox blood disappear protein B SA, nucleic acid dye GoodView (Beijing Suo Laibao), distilled water (worker is given birth in Shanghai), it is synthetic that primer is given birth to the worker by Shanghai.
Polymerase chain reaction (50 μ L system) reagent: contain 10 times of polymerase chain reaction damping fluids and (contain MgCl
2), special primer is 10 μ M, dNTP (2.5mM each), bovin serum albumin BSA (5mg/ml) and the mattress distilled water that goes out, EXTaq enzyme.
The composition of 10 times of PCR Buffer is as follows:
Tris-HCl 100mM
KCl 500mM
MgCl
2 15mM。
A kind of multiple PCR reagent kit that detects mosquito biography pathogenic agent of the present invention, contain following primer:
1) primer of detection flavivirus first round PCR
The sequence of upstream primer shown in SEQ ID NO:1,
The sequence of downstream primer is shown in SEQ ID NO:2;
2) detect the primer that flavivirus second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:3,
The sequence of downstream primer is shown in SEQ ID NO:4;
3) primer of detection plasmodium first round PCR
The sequence of upstream primer shown in SEQ ID NO:5,
The sequence of downstream primer is shown in SEQ ID NO:6;
4) detect the primer that plasmodium second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:7 or shown in SEQ ID NO:8,
The sequence of downstream primer is shown in SEQ ID NO:9;
5) primer of detection Wuchereria malayi first round PCR
The sequence of upstream primer shown in SEQ ID NO:10,
The sequence of downstream primer is shown in SEQ ID NO:11;
6) detect the primer that Wuchereria malayi second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:12,
The sequence of downstream primer is shown in SEQ ID NO:13.
Further, described flavivirus comprises encephalitis b virus or dengue fever virus 1 type or dengue fever virus 2 types or dengue fever virus 3 types or dengue fever virus 4 types, west nile virus and yellow fever virus.
Further, described plasmodium comprises plasmodium falciparum or Plasmodium vivax or Plasmodium knowlesi or Plasmodium ovale or malariae.
Further, also contain polymerase chain reaction reagent in the described test kit.
Further, also contain negative quality control standard product in the described test kit.
Further, also contain the positive criteria product in the described test kit.
Further, described negative quality control standard product are sterilization ddH
2O.
Further, described positive criteria product are encephalitis B cDNA or dengue fever virus cDNA or Plasmodium vivax DNA or plasmodium falciparum DNA.
Further, each component is in the reagent of described polymerase chain reaction:
1) 10 times of polymerase chain reaction Huan Red liquid;
2) four kinds of deoxyribonucleotides, the concentration of four kinds of deoxyribonucleotides is 2.5mM;
3) bovin serum albumin BSA, the concentration of bovin serum albumin BSA is 5mg/ml;
4) EXTaq enzyme, the concentration of EXTaq enzyme are 5U/ μ L;
5) sterilization distilled water.
Further, the composition of described 10 times of polymerase chain reaction Huan Red liquid is as follows:
Tris-HCl 100mM
KCl 500mM
MgCl
2 15mM。
Further, the concentration of all primers in the described test kit is 10 μ M.
The present invention also provides a kind of method that mosquito passes pathogenic agent that detects, comprise a step of extracting mosquito nucleic acid, also comprise a step that the nucleic acid that extracts is increased on the pcr amplification instrument, comprise that also a product with amplification carries out the step of electrophoresis detection, also comprise and determine that mosquito passes the order-checking step of the concrete kind of pathogenic agent.
Further, in a step that the nucleic acid that extracts is increased on the pcr amplification instrument,
1) primer of detection flavivirus first round PCR
The sequence of upstream primer shown in SEQ ID NO:1,
The sequence of downstream primer is shown in SEQ ID NO:2;
2) detect the primer that flavivirus second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:3,
The sequence of downstream primer is shown in SEQ ID NO:4;
3) primer of detection plasmodium first round PCR
The sequence of upstream primer shown in SEQ ID NO:5,
The sequence of downstream primer is shown in SEQ ID NO:6;
4) detect the primer that plasmodium second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:7 or shown in SEQ ID NO:8,
The sequence of downstream primer is shown in SEQ ID NO:9;
5) primer of detection Wuchereria malayi first round PCR
The sequence of upstream primer shown in SEQ ID NO:10,
The sequence of downstream primer is shown in SEQ ID NO:11;
6) detect the primer that Wuchereria malayi second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:12,
The sequence of downstream primer is shown in SEQ ID NO:13.
A. extract nucleic acid: get single mosquito (anopheles stephensi) and put into the 1.5ml centrifuge tube and be ground to it and be crushed into powder fully, use a day root DNA extraction test kit DP304 (sky, Beijing root), extract DNA by explanation.
The b.PCR amplification:
First round PCR condition
10×Buffer 5μL
dNTP(2.5mM) 4μL
BSA(5mg/ml) 1μL
Each 1 μ L (filaria plasmodium first round PCR primer) of FP (10 μ M)
Each 1 μ L (filaria plasmodium first round PCR primer) of RP (10 μ M)
Dna profiling 1.5 μ L
EXTaq 0.4μL(2U)
ddH
2O 34.1μL
The PCR loop parameter
1.?94℃ 4Min
2.?94℃ 25Sec
3.?56℃ 20Sec
4.?72℃ 50Sec
5.?go?to?step?2 29-33times
6.72℃ 5Min
Second takes turns the PCR condition
10×Buffer 5μL
dNTP(25mM) 4μL
BSA(5mg/ml) 1μL
Each 1 μ L (filaria plasmodium second is taken turns the PCR primer) of FP (10 μ M)
Each 1 μ L (filaria plasmodium second is taken turns the PCR primer) of RP (10 μ M)
Dna profiling (first round PCR product) 1 μ L
EXTaq 0.4μL(2U)
ddH
2O 34.6μL
total 50μL
The PCR loop parameter
1.?94℃ 4Min
2. 94℃ 25?Sec
3. 56℃ 20?Sec
4. 72℃ 35?Sec
5. go?to?step?2 34times
6. 72℃ 5Min
Primer recited above is specially:
The first round PCR primer of plasmodium
Plasmodium special primer NUE937-957E:5 '-AGCTTGGGGACATTTGTATTCA-3 '
Plasmodium special primer NUE1707-1688E:5 '-GCGTGCAGCCTAGTTCATCT-3 '
Second of plasmodium is taken turns the PCR primer
Plasmodium special primer NUE1199-1216E:5 '-GGGTTCTGGGGCGAGTAT-3 ' or plasmodium special primer NUE1199-1218E:5 '-GGGTTCTGGGGCGAGTATTC-3 '
Plasmodium special primer NUE1707-1688E:5 '-GCGTGCAGCCTAGTTCATCT-3 '
The first round PCR primer of Wuchereria malayi
Wuchereria malayi special primer ML8189-8206E:5 '-AGGCGTTGTTTGATGGTC-3 '
Wuchereria malayi special primer ML8619-8602E:5 '-GAAAGGGGCACGATGTAA-3 '
Second of Wuchereria malayi is taken turns the PCR primer
Wuchereria malayi special primer ML8440-8458E:5 '-GGTGGTTTACGGTCTTGTG-3 '
Wuchereria malayi special primer ML8619-8602E:5 '-GAAAGGGGCACGATGTAA-3 '
C. electrophoresis and observation: the sepharose of preparation 1.5-2%, get the sample after the 5-10 μ L amplification, under ultraviolet lamp, observe, detect whether a 180bp is arranged, if the band of a 180bp is arranged, illustrate that mosquito carries Wuchereria malayi, otherwise then do not have; If have the band explanation mosquito of an about 508bp to carry plasmodium (plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium ovale, malariae), otherwise then do not have.If will determine the plasmodium kind, the rubber tapping purifying is taken turns the PCR upstream primer with second and is checked order (this easily success of segment order-checking), infects the situation that should not directly check order for multiple plasmodium, but the cloning and sequencing solution.As shown in Figure 1.
Detect the following description of step that mosquito is carried flavivirus (encephalitis b virus, dengue fever virus 1-4 type, yellow fever virus):
A. extract nucleic acid: get mosquito (anopheles stephensi) virus-culturing fluid, use a day root DNA/RNA to extract test kit DP422 altogether, extracting DNA/RNA by explanation, is cDNA with the Quant cDNA first chain synthetic agent (the KR103cDNA first chain synthetic agent box) with the RNA reverse transcription.
The b.PCR amplification:
Detect flavivirus (encephalitis b virus, dengue fever virus 1-4 type, yellow fever virus)
First round PCR condition
10×Buffer 5μL
dNTP(2.5mM) 4μL
BSA(5mg/ml) 1μL
FP：malm(10μM) 1μL
RP：Rcfm9279-9301(10μM) 1μL
CDNA template 1.0 μ L
EXTaq 0.3μL(1.5U)
ddH2O 36.7μL
The PCR loop parameter
1. 94℃ 4Min
2. 94℃ 25Sec
3. 53℃ 25Sec
4. 72℃ 30Sec
5. go?to?step?2 25-30times
6. 72℃ 5Min
Second takes turns the PCR condition
10×Buffer 5μL
dNTP(25mM) 4μL
BSA(5mg/ml) 1μL
FP：R9102-9123(10μM) 1μL
RP：Rcfm9279-9301(10μM) 1μL
Template (first round PCR product) 1 μ L
EXTaq 0.3μL(1.5U)
ddH
2O 36.7μL
total 50μL
The PCR loop parameter
1. 94℃ 4Min
2. 94℃ 25Sec
3. 53℃ 20Sec
4. 72℃ 30Sec
5. go?to?step?2 32-34times
6. 72℃ 5Min
Primer recited above is specially:
The first round PCR primer of flavivirus
Special primer malm:5 '-AACATGTTGGGRAARAGAGAGAA-3 '
Special primer Rcfm9279-9301:5 '-GTGTCCCAKCCKGCKGTRTCATC-3 '
Second of flavivirus is taken turns the PCR primer
Special primer R9102-9123:5 '-ATYTGGTWYATGTGGYTTGGAG-3 '
Special primer Rcfm9279-9301:5 '-GTGTCCCAKCCKGCKGTRTCATC-3 '
C. electrophoresis and observation: the sepharose of preparation 1.5-2%, get the sample after 5-10 μ L increases, under ultraviolet lamp, observe, detect the nucleic acid band whether a treaty 200bp is arranged, if the nucleic acid band of a 200bp is arranged, illustrate that mosquito carries flavivirus (encephalitis b virus, dengue fever virus 1-4 type, yellow fever virus), otherwise then do not have.If will determine the type of flavivirus, the rubber tapping purifying is taken turns the PCR upstream primer with second and is checked order (this easily success of segment order-checking), infect the situation that should not directly check order simultaneously for multiple flavivirus, but cloning and sequencing solves.As shown in Figure 2.
Claims (14)
1. one kind is detected the multiple PCR reagent kit that mosquito passes pathogenic agent, it is characterized in that containing following primer:
1) primer of detection flavivirus first round PCR
The sequence of upstream primer shown in SEQ ID NO:1,
The sequence of downstream primer is shown in SEQ ID NO:2;
2) detect the primer that flavivirus second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:3,
The sequence of downstream primer is shown in SEQ ID NO:4;
3) primer of detection plasmodium first round PCR
The sequence of upstream primer shown in SEQ ID NO:5,
The sequence of downstream primer is shown in SEQ ID NO:6;
4) detect the primer that plasmodium second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:7 or shown in SEQ ID NO:8,
The sequence of downstream primer is shown in SEQ ID NO:9;
5) primer of detection Wuchereria malayi first round PCR
The sequence of upstream primer shown in SEQ ID NO:10,
The sequence of downstream primer is shown in SEQ ID NO:11;
6) detect the primer that Wuchereria malayi second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:12,
The sequence of downstream primer is shown in SEQ ID NO:13.
2. a kind of multiple PCR reagent kit that mosquito passes pathogenic agent that detects as claimed in claim 1, it is characterized in that: described flavivirus comprises encephalitis b virus or dengue fever virus 1 type or dengue fever virus 2 types or dengue fever virus 3 types or dengue fever virus 4 types, west nile virus and yellow fever virus.
3. a kind of multiple PCR reagent kit that mosquito passes pathogenic agent that detects as claimed in claim 1, it is characterized in that: described plasmodium comprises plasmodium falciparum or Plasmodium vivax or Plasmodium knowlesi or Plasmodium ovale or malariae.
4. a kind of multiple PCR reagent kit that mosquito passes pathogenic agent that detects as claimed in claim 1 is characterized in that: also contain polymerase chain reaction reagent in the described test kit.
5. a kind of multiple PCR reagent kit that mosquito passes pathogenic agent that detects as claimed in claim 1 is characterized in that: also contain negative quality control standard product in the described test kit.
6. a kind of multiple PCR reagent kit that mosquito passes pathogenic agent that detects as claimed in claim 1 is characterized in that: also contain the positive criteria product in the described test kit.
7. a kind of multiple PCR reagent kit that mosquito passes pathogenic agent that detects as claimed in claim 5 is characterized in that: described negative quality control standard product are sterilization ddH
2O.
8. a kind of multiple PCR reagent kit that mosquito passes pathogenic agent that detects as claimed in claim 6 is characterized in that: described positive criteria product are encephalitis B cDNA or dengue fever virus cDNA, Plasmodium vivax DNA, plasmodium falciparum DNA.
9. a kind of multiple PCR reagent kit that mosquito passes pathogenic agent that detects as claimed in claim 4 is characterized in that each component is in the reagent of described polymerase chain reaction:
1) 10 times of polymerase chain reaction Huan Red liquid;
2) four kinds of deoxyribonucleotides, the concentration of four kinds of deoxyribonucleotides is 2.5mM;
3) bovin serum albumin BSA, the concentration of bovin serum albumin BSA is 5mg/ml;
4) EXTaq enzyme, the concentration of EXTaq enzyme are 5U/ μ L;
5) sterilization distilled water.
10. a kind of multiple PCR reagent kit that mosquito passes pathogenic agent that detects as claimed in claim 9 is characterized in that the composition of described 10 times of polymerase chain reaction Huan Red liquid is as follows:
Tris-HCl 100mM
KCl 500mM
MgCl
2 15mM。
11. a kind of multiple PCR reagent kit that mosquito passes pathogenic agent that detects as claimed in claim 1, it is characterized in that: the concentration of all primers in the described test kit is 10 μ M.
12. one kind is detected the method that mosquito passes pathogenic agent, it is characterized in that: comprise a step of extracting mosquito nucleic acid, also comprise a step that the nucleic acid that extracts is increased on the pcr amplification instrument, comprise that also a product with amplification carries out the step of electrophoresis detection, also comprise and determine that mosquito passes the order-checking step of the concrete kind of pathogenic agent.
13. a kind of method that mosquito passes pathogenic agent that detects as claimed in claim 12 is characterized in that:
In a step that the nucleic acid that extracts is increased on the pcr amplification instrument,
1) primer of detection flavivirus first round PCR
The sequence of upstream primer shown in SEQ ID NO:1,
The sequence of downstream primer is shown in SEQ ID NO:2;
2) detect the primer that flavivirus second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:3,
The sequence of downstream primer is shown in SEQ ID NO:4;
3) primer of detection plasmodium first round PCR
The sequence of upstream primer shown in SEQ ID NO:5,
The sequence of downstream primer is shown in SEQ ID NO:6;
4) detect the primer that plasmodium second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:7 or shown in SEQ ID NO:8,
The sequence of downstream primer is shown in SEQ ID NO:9;
5) primer of detection Wuchereria malayi first round PCR
The sequence of upstream primer shown in SEQ ID NO:10,
The sequence of downstream primer is shown in SEQ ID NO:11;
6) detect the primer that Wuchereria malayi second is taken turns PCR
The sequence of upstream primer shown in SEQ ID NO:12,
The sequence of downstream primer is shown in SEQ ID NO:13.
14. the described a kind of application of multiple PCR reagent kit in detecting encephalitis b virus or dengue fever virus 1 type or dengue fever virus 2 types or dengue fever virus 3 types or dengue fever virus 4 types or west nile virus and yellow fever virus/plasmodium falciparum or Plasmodium vivax or Plasmodium knowlesi or Plasmodium ovale or malariae/Wuchereria malayi or bancroft's filaria that mosquito passes pathogenic agent that detect of claim 1.
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| CN102534048A (en) * | 2012-01-11 | 2012-07-04 | 中国检验检疫科学研究院 | Non-diagnostic method for detecting flavivirus by TaqMan probe through real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) |
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| CN103981282A (en) * | 2014-04-25 | 2014-08-13 | 成都军区疾病预防控制中心军事医学研究所 | Method for collecting, preserving and detecting mosquito-borne viruses under normal temperature |
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| CN102534048A (en) * | 2012-01-11 | 2012-07-04 | 中国检验检疫科学研究院 | Non-diagnostic method for detecting flavivirus by TaqMan probe through real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) |
| CN102534048B (en) * | 2012-01-11 | 2013-11-27 | 中国检验检疫科学研究院 | Non-diagnostic method for detecting flavivirus by TaqMprobe through real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) |
| CN102586480A (en) * | 2012-02-24 | 2012-07-18 | 中国检验检疫科学研究院 | Non-diagnostic method for detecting flavivirus and alphavirus through double TaqMan probe real-time fluorescence RT-PCR |
| CN103981282A (en) * | 2014-04-25 | 2014-08-13 | 成都军区疾病预防控制中心军事医学研究所 | Method for collecting, preserving and detecting mosquito-borne viruses under normal temperature |
| CN103981282B (en) * | 2014-04-25 | 2016-01-13 | 成都军区疾病预防控制中心军事医学研究所 | The collection of carapuru virus normal temperature, preservation and detection method |
| CN104673919A (en) * | 2015-02-27 | 2015-06-03 | 中国疾病预防控制中心寄生虫病预防控制所 | Kit and method for identifying species of human malaria parasites |
| CN106047993A (en) * | 2016-04-20 | 2016-10-26 | 金弗康生物科技(上海)有限公司 | Molecular markers for five important pathogens and application thereof |
| CN111286557A (en) * | 2020-01-16 | 2020-06-16 | 山西国际旅行卫生保健中心(太原海关口岸门诊部) | Reagent combination, kit and application for detecting blood transfusion sexually transmitted pathogen |
| CN111286557B (en) * | 2020-01-16 | 2023-12-08 | 山西国际旅行卫生保健中心(太原海关口岸门诊部) | Reagent combination for detecting haemostatic transmission pathogen, kit and application |
| CN111763772A (en) * | 2020-08-13 | 2020-10-13 | 中国医学科学院输血研究所 | Premixed fluorescent PCR (polymerase chain reaction) detection reagent and kit for mosquito-borne infectious disease pathogens |
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Application publication date: 20110223 |