| PART IA sequencing technique for RHCE gene coding regionBackground:Rh blood group system includes more than 50 antigens, closely related with clinical antigens are mainly D, C, c, E and e antigen.In addition to the D antigen, RhC, c, E and e antigen can cause hemolytic disease of the fetal newborn and delayed hemolytic transfusion reaction.Recently irregular antibodies induced by transfusion in long-term of allogeneic transfusion patient has gained more attention, especially in Rh blood group system associated antibody (anti-E>anti-C>anti-e>anti-c).This antibody will seriously affect the patient clinical blood transfusion safety and effectiveness.Correct blood type is the precondition of matched blood transfusion. Currently the main methods for clinical blood typing is still the serological method, serological methods have the following shortcomings in the actual application process:the existence of various cell populations or chimeric phenomenon, such as long-term and regular blood transfusion,after hematopoietic stem cell transplantation, mixed agglutination caused by injection of ABO incompatible blood; direct antibody positive individuals, because of its high price the presence of self antibodies,antigen-antibody reaction can occur strong with all red blood cells in vitro; weak expression of antigen, due to the decrease number of antigen molecules in RBC membrane, the antigen-antibody reaction or serological detection results weakened, were always identified as negative; and other clinical difficulties such as red blood cells insufficient or missing etc..The development of PCR technology takes the blood type research into the molecular level.Currently the most commonly used PCR genotyping technology is sequence specific primer PCR (PCR-SSP) at home and abroad, PCR-SSP,a specific DNA sequence polymorphism analysis technique,its basic principle is designing a sequence specific primers according to differences in the nucleotide sequence and this pair of primers can only amplify the complementary pairing purpose sequence, at last according to whether there is a amplification product todetermine genotype.With the advantages of low cost, simple operation and strong specificity etc., PCR-SSP technology has been widely used in blood group genetyping. Studies have shown that the molecular mechanism of RhC/c and RhE/e polymorphism,7 base mutation, which is the theoretical source of PCR-SSP genotype.RhC/c refers to 1,2 of the RHCE gene exon,contains six base substitutions (48G>C,150C> T,178C> A, 201A>G,203A> G and 307C>T) and resulted in 4 amino acid substitutions (Cysl6Trp, ILe60Leu, Ser68Asn and Ser103Pro, involving second extracellular loop; the RhE/e antigen refers to exon 5,1 base substitutions (C>G) led to the replacement of 1 amino acids (Pro226 Ala), involving fourth extracellular loop.PCR-SSP used in RHCE genotyping is also have some obvious shortcomings,1) PCR-SSP method exists a certain proportion of false positive or false negative results, because of the 48th base mutation in exon 1 and 109 bp insertion deletion in intron 2, RHC allele detection will lead to false positive and false negative results respectively. Because of 178,201,203 and (or) 307 sites mutation in exon 2,RHc allele detection will be inaccurate.2) polymorphism of RHCE allele, the detection of RHCE alleles has more than 70, but the common genotyping primers did not detect all the RHCE allele.3)complex genetic factors, different nationalities and different populations of RH gene has a different genetic background, such as RhD Negative phenotype frequencies among Caucasian populations are more than 15%, but Asians is far lower less 1%. At the same time, the Caucasian has more RHD and RHCE gene rare mutation type, which not only includes all nucleotide substitution, insertion and deletion, also include more complex RHD/RHCE gene exchange. These complex variations that PCR-SSP often appear false positive or false negative in typing.Sequencing method, especially with the development of second-generation sequencing technology, More simple,more efficient and cheaper are the advantages of second-generation sequencing technology,sequencing has become a new genotyping and sequence analysis techniques following the PCR-SSP technique.Previous reports only concern one or a few exon sequence analysis, not refer to the whole RHCE gene coding region. In this paper, we design a simple RHCE gene coding region of full-length sequencing method, and apply it to the study of chronically transfused patients, RHCE gene mutation individual and family investigation, and RhCE blood group systemmatching blood transfusion.Aims:This study aimed to establish a sequencing technique for RHCE gene coding region, and and it is convenient for clinical laboratory RHCE genotyping.Methods:l.The research object and material:400 blood donor samples collected in Shenzhen blood center,Shenzhen,China.A D--sample from shanxi province blood center,1 case of Rh negative samples from the in Shenzhen blood center, and 1 cases of weak E samples from Liaoning Province Blood Center.2. The research methods:(1) Serological tests:Anti-RhC, anti-Rhc,anti-RhE and anti-Rhe were used to identify RBC surface RhCcEe antigen phenotype.(2) Genotyping by PCR-SSP:according to gene sequence differences between the RHCE and RHD gene,designing RHC, RHc, RHE and RHe four amplification system. Baesd on amplification products appearance and not to determine the genotypes.(3) Sequencing:according to the specificity of the RHCE gene sequence,10 pairs of specific primers were designed to amplify 10 exon sequences of the RHCE gene, amplification products confirmed by electrophoresis, and then the PCR products were purified and sequencing.Finally comparison analysis of the gene sequence obtained.Results:1.The 400 blood donation sample serological tests results show that it contain nine phenotypes, and from 400 blood donation sample screened7 cases of weak expression samples.2.The genotyping results of 400 cases of blood donation samples (including 7 cases of weak expression sample) by PCR-SSP method was consistent with the serological;D-- sample results show:his RHCE genotype is DC-, and does not match serological typing results (D--),and consistent with serological typing results. Rh negative sample genotyping results (dccee) also consistent with serological results.3.The sequencing results showed:the RHCE gene sequences of 399 cases of blood donation sample in 400 cases(including 6 cases of weakly expressed samples) are consistent with normal RHCE gene sequences.1 case phenotype Ccee (weak c) sample exists a mutation,namely 1059GA (GenBank accession number:KT957625) and it lead to replacement of amino acid W353*. D--sequencing results:PCR amplification and sequencing results show that proband and younger brother were missing 3th,4th and 5th exons, and the other exon sequencing results did not find variation. The PCR-SSP typing results showed RHC positive (DC-). Weak e samples observed the has a mutation (762G>C). To sum up, the proband and brother sample sequencing results show that carry a new allel formed by RHCE/RHD exchange,namelyRHCE-D (3-5)CE.Conclusion:1.TheRHCE gene coding region of full-length sequencing method can under the reaction conditions of the consent of the successful amplification specificity of the RHCE gene with 10 exons.2.TheRHCE gene coding region of full-length sequencing method for general laboratory RHCE gene sequencing and solve clinical difficult to finalize the design of the sample;3.TheRHCE gene coding region of full-length sequencing method for long-term patients with massive blood transfusion and difficult type sample of accurate shaping and matching blood transfusion has important significance, correct setting is the premise of the safety of blood transfusion, but also to reduce the Alloantibody produced an important means.PART IIA method for detecting blood borne malariaAt present, the incidence of malaria in China is decreasing year by year, but with the economic and trade exchanges between China and Southeast Asia, Africa, South America and other areas of high incidence of malaria, malaria prevention and control situation is still grim. According to the latest information released by the WHO, in 2013 there were still a total of about 198,000,000 malaria patients, of which there are more than 584,000 person died of malaria.Since Woolsey reported the first case of transfusion malaria in 1911, there have been reports of blood borne malaria in the world. Blood transfusion malaria occur from time to time because of many countries haven’t carry out the project of malaria detection. This not only makes the patient the original condition is more serious,but also makes clinical blood transfusion safety face more risk. In recent years,immigration and repatriation of malaria cases and deaths found in Beijing, Guangdong, Zhejiang, Jiangsu, Sichuan and other places one after another.The risk of transfusion malaria infection is gradually increased in our country.The parasites infecting human have 5 kinds, they are P. falciparum, p.vivax, P. malariae,p.ovale and p.knowlesi respectively, which P. falciparum is the most common falciparum and has the highest death rate. The female and male of Plasmodium parasites grow up in mosquito and invade the human body through bite. Plasmodium digest the globin moiety of hemoglobin as source of nutrients in the host red blood cell, and heme of hemoglobin release into free heme, in order to avoid killing effects of free heme, the parasite transform free heme into insoluble hemin crystals,namely hemozoin (also is known as malaria pigment) . Hemozoin is synthesized in the food vacuole of Plasmodium, and released into the plasma accompany with plasmodium infected red blood cell(iRBCs) lysis, most of them are swallowed up by phagocytes. It is seen that hemozoin appeared in the red blood cells, serum and phagocytes of malaria patients.At present, the existing detection methods of malaria can be divided into three categories,the first is the blood smear,Giemsa stained blood smear for optical microscope to observe whether there is Plasmodium as the judgment standard, this method is still the "gold standard" for detection of malaria.But there are time-consuming,labor-intensive and interpretation requires considerable expertise, the second is the antigen and antibody detection, researchers developing many kinds of detection methods based on the principle of antigen-antibody reaction, such as enzyme linked immunosorbent assay(ELISA), immune colloidal gold chromatography, electrochemical luminescence technique(ECL),this methods are operation fast, but there have a certain percentage of false negative results and the degree of sensitivity is poor.The third is the nucleic acid detection,PCR is the representative technology for detection of malaria specific nucleic acid fragments.And the sensitivity of PCR is greatly improved but high experiment requires limit its widely use.Raman spectroscopy is a comprehensive cross technology, involving optical physics, material science, computer scienceand so on. Its basic principle is:a beam of monochromatic light irradiated medium will appear transmission, absorption or scattering, most of thescattered light wavelength is the same as incident light, known as Rayleigh scattering. And a small part of light through the media and molecular interaction of the frequency changed by the scattering,called Raman scattering.Through the analysis of Raman spectrum peak position, intensity and width of molecular vibration and rotation of the information, which can reflect chemical bonds or different functional groups in the molecule.In a word, Raman spectroscopy is an effective method to research the the molecular structure of material. Raman spectroscopy has the following two advantages, one is nondestructive, Raman spectroscopy is a new technique for nondestructive detection, the research object in the field of life science are proteins, carbohydrates, lipids and nucleic acid. Raman laser photon energy is low and does not produce ionization damage, which is especially suitable for detecting cells and organization. The sample does not need to stain or remove impurities. Liquid, solid and even living cells can also be detected in situ, which is no impact on the sample after detection. That can avoid sample loss and physical properties change in the process of detection. The other is specific spectra, each substance has characteristic fingerprint Raman spectra, namely characteristic peaks of Raman spectrum.Raman spectroscopy is especially sensitive to the biological macromolecules such as nucleic acids, proteins, sugars, lipids and so on. And it is particularly sensitive to detect subtle changes in the structure of the material. It has advantages in analyzing the composition and structure of the biological macromolecules in the physiological and (or) pathological state.Aims:The purpose of this study is to establish a platform for Raman spectrum detection with high sensitivity and high specificity, provides a new method for the accurate, simpleand sensitive for detection of malaria.Methods:1 research objects and materials:Sources of experimental samples on the in vitro synthesis of beta-hemoglobin, Plasmodium falciparum in vitro red blood cells culture, Plasmodium falciparum erythrocyte in vitro culture under different insect blood rate (10%,5%,2.5%,1%,0.5% and 0.01%) malaria pigment extract and normal uninfected fresh red cells.2 experimental methods:(1) The culture of P. falciparum:Plasmodium falciparum line 3D7, using 37℃and 5%CO2 in continuous culture.(2) The P-hematin synthesis and Raman spectroscopy detection:p-hematin synthetic method is acid extraction, β-hematin powder uniform smear on a glass slide and then placed in shot detection. The detection instrument is the confocal-micro Raman spectrometer.(3) Hemozoin extraction and Raman spectroscopy detection:hemozoin detection conclue hemozoin extracts detection and hemozoin detection in situ, and hemozoin extracted using saponin solution lysis host red blood cells to release hemzoin. Confocal-micro Raman spectrometer was used for hemozoin extracts detection and fast imaging system for iRBC detection in situ.(4) The application of SERS in detection of hemozoin:two different surface enhanced materials are used to enhance the Raman signal of hemozoin.Results:1.The Raman spectra of the synthesized β-hematin has a characteristic peak and it is consistent with hemozoin.2.The iRBC and nRBC blood smear detection results show that the Raman spectra of the relative strength of the coefficient of variation is less than 20% and the coefficient of variation of relative displacement is less than 1%, both of which have good consistency, microscopic Raman spectrum detection can not distinguish normal individuals and Plasmodium infected individuals using blood smear.3.The method of iRBC malaria pigment extraction was established. Raman spectroscopy can effectively identify the malaria pigment and hemoglobin, the detection infection rate is reach to 0.1%.4.RAMAN-11 rapid Raman scanning imaging system was employed to scanning suspicious iRBC and nRBC. Taking 1372cm-1 Raman peak as a reference peak, we made the hemozoin distribution map that consistent with hemozoin space distribution of the light microscope, namely Plasmodium in iRBC in situ scanning imaging and optical microscope of malaria pigment spatial distribution has a good consistency.5.Screening out the signal amplification effect of the surface enhanced substrate. It can achieve the purpose of signal amplification and better protection of the sample.Conclusion:1.Raman spectroscopy can be used for detection of Plasmodium, successfully getting the characteristic peaks of malaria pigment, and successfully identifying iRBC and nRBC.2.Raman spectra of hemozoin extraction and β-hemoglobin (positive control) has a good consistency, but beween hemozoin extraction and normal hemoglobin (negative control) there is a marked difference, concentrate hemozoin extraction can make the detection sensitivity to 0.1% parasitemia.3.Clear malaria pigment distribution map can be seen in the iRBC scanning mapping in situ. And it can used to identifying food vacuole and insect body, detecting hemozoin position and measuring the relative content of malaria pigment. |
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