RESUMEN

Fatal gout in geese caused by goose astrovirus (GAstV) has been spreading rapidly in China since 2018, causing serious economic losses in the goose breeding industry. To achieve simple, convenient and sensitive detection of GAstV, a novel diagnostic test was developed by combining reverse transcription-enzymatic recombinase amplification (RT-ERA) and CRISPR-Cas12a technologies. RT-ERA primers were designed to pre-amplify the conserved region of the ORF2 gene of GAstV and the predefined target sequence detected using the Cas12a/crRNA complex at 37℃ for 30 min. Specific detection of GAstV was achieved with no cross-reaction with non-GAstV templates and a sensitivity detection limit of 2 copies. The experimental procedure could be completed within 1 h, including RNA extraction (15 min), RT-ERA reaction (20 min), CRISPR-Cas12a/crRNA detection (5 min) and result readout (within 2 min) steps. In conclusion, the combination of RT-ETA and CRISPR-Cas12a provides a rapid and specific method that should be effective for the control and surveillance of GAstV infections in farms from remote locations.

Asunto(s)

Avastrovirus , Transcripción Reversa , Animales , Recombinasas , Gansos/genética , Sistemas CRISPR-Cas , Pollos , Avastrovirus/genética

RESUMEN

Since the introduction of the polymerase chain reaction (PCR) technique in 1983, nucleic acid amplification has permeated all fields of biological science, particularly clinical research. Despite its importance, PCR has been restricted to specialized centers and its use in laboratories with few resources is limited. In recent decades, there has been a notable increase in the development of new isothermal technologies for molecular diagnosis with the hope of overcoming the traditional limitations of the laboratory. Among these technologies, recombinase polymerase amplification (RPA) has a wide application potential because it does not require thermocyclers and has high sensitivity, specificity, simplicity, and detection speed. This technique has been used for DNA and RNA amplification in various pathogenic organisms such as viruses, bacteria, and parasites. In addition, RPA has been successfully implemented in different detection strategies, making it a promising alternative for performing diagnoses in environments with scarce resources and a high burden of infectious diseases. In this study, we present a review of the use of RPA in clinical settings and its implementation in various research areas.

Asunto(s)

Técnicas de Amplificación de Ácido Nucleico , Recombinasas , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , Recombinasas/genética , Sensibilidad y Especificidad

RESUMEN

Familial colorectal cancer type X (FCCTX) is a heterogeneous colorectal cancer predisposition syndrome that, although displays a cancer pattern similar to Lynch syndrome, is mismatch repair proficient and does not exhibit microsatellite instability. Besides, its genetic etiology remains to be elucidated. In this study we performed germline exome sequencing of 39 cancer-affected patients from 34 families at risk for FCCTX. Variant classification followed the American College of Medical Genetics and Genomics (ACMG) guidelines. Pathogenic/likely pathogenic variants were identified in 17.65% of the families. Rare and potentially pathogenic alterations were identified in known hereditary cancer genes (CHEK2), in putative FCCTX candidate genes (OGG1 and FAN1) and in other cancer-related genes such as ATR, ASXL1, PARK2, SLX4 and TREX1. This study provides novel important clues that can contribute to the understanding of FCCTX genetic basis.

Asunto(s)

Quinasa de Punto de Control 2/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , ADN Glicosilasas/genética , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Enzimas Multifuncionales/genética , Adulto , Anciano , Proteínas de la Ataxia Telangiectasia Mutada/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Reparación de la Incompatibilidad de ADN/genética , Femenino , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal/genética , Humanos , Masculino , Persona de Mediana Edad , Oncogenes/genética , Fosfoproteínas/genética , Recombinasas/genética , Proteínas Represoras/genética , Ubiquitina-Proteína Ligasas/genética , Secuenciación del Exoma

RESUMEN

Burkholderia pseudomallei causes a fatal and infectious disease, melioidosis or Whitmore's disease in humans and animals. Melioidosis is present in different parts of the world and is endemic in Southeast Asia and Northern Australia. Accurate diagnosis of melioidosis is difficult due to its common flu-like symptoms, potentially long incubation period and erroneous identification as culture contaminant. Early diagnosis of the disease is essentially required for administration of suitable antibiotics and disease containment. The present study reports a rapid, specific and sensitive recombinase polymerase amplification lateral flow assay for detection of B. pseudomallei. Specific primers and probe were designed and the assay was performed at 41 °C for 20 min in a portable incubator. End products were detected using ready-to-use lateral flow strips. RPA lateral flow assay could detect ≥ 250 fg genomic DNA of B. pseudomallei and ≥ 50 copies of recombinant plasmid harbouring the target DNA sequence. The assay was found to be highly specific and did not cross-react with other bacterial strains. In artificially spiked human blood and urine samples, the detection limit of the assay was 4.8 × 104 and 4.95 × 104 CFU/mL of B. pseudomallei, respectively. The detection limit of assay after 6 h of enrichment of artificially spiked urine samples was found to be 4.95 × 103 CFU/mL of B. pseudomallei. Detection limit in artificially spiked tap water and soil samples was determined to be 7.5 × 102 CFU/mL and 3.3 × 104 CFU per 5 g of B. pseudomallei, respectively.

Asunto(s)

Burkholderia pseudomallei , Melioidosis , Animales , Burkholderia pseudomallei/genética , Cartilla de ADN/genética , Humanos , Melioidosis/diagnóstico , Melioidosis/microbiología , Recombinasas

RESUMEN

Cutaneous leishmaniasis (CL) is highly prevalent in rural and sylvatic regions of Latin America, with an estimated 55,000 annual cases. Diagnosis in resource-limited areas still relies on microscopy of dermal scrapings, while more sensitive methods like PCR are not attainable due to costs and lack of adequate health infrastructure. Isothermal amplification of Leishmania DNA can be performed without sophisticated equipment and training and may become a point of care (POC) test for health care centers with scarce resources. We evaluated the efficacy of recombinase-polymerase-amplification (RPA-LF) to diagnose CL in 226 patients attending a clinic in Puerto Maldonado within the Peruvian Amazon basin. Conventional PCR targeting kinetoplast DNA (kDNA-PCR) was used as the gold standard. Eight of 226 patients were considered true negatives (microscopy, kDNA-PCR, and RPA-LF negative), while RPA-LF resulted positive in 186 of 204 kDNA-PCR positive patients, yielding 91.2% (confidence interval [CI] = 86.5-94.4%) sensitivity and 93% (CI 88.6-95.8%) positive predictive value. There were 14% (32/226) discrepant samples alternating positive and negative results in similar proportions between both tests. Quantitative PCR used to resolve the discrepancies suggested that they occurred in samples with scarce parasite numbers as determined by high cycle threshold (Ct) values (≥32; cutoff 35.5). Microscopy had the lowest sensitivity of all methods (45.4%). Nested real-time PCR performed in 71 samples determined that Leishmania (Viannia) braziliensis was highly prevalent (69/71), and Leishmania (Viannia) lainsoni was present in only two isolates. Results indicated that RPA-LF has POC potential for CL endemic areas, yet further simplification and optimization coupled with field validation will be necessary to confirm its broad applicability.

Asunto(s)

Leishmaniasis Cutánea , Recombinasas , Animales , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/epidemiología , Leishmaniasis Cutánea/veterinaria , Perú/epidemiología , Bosque Lluvioso , Lectura , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Sensibilidad y Especificidad

RESUMEN

BACKGROUND: The diagnosis of cutaneous leishmaniasis (CL) is based on demonstration of the presence of the parasite in samples obtained from the lesions by direct examination (DE), culture or polymerase chain reaction (PCR)-based molecular tests. Recombinase polymerase amplification (RPA) represents an isothermal version of the conventional PCR (cPCR) technique, being ideal for detecting Leishmania DNA, especially in field conditions. METHODS: A prospective and cross-sectional study was conducted to evaluate the diagnostic performance of RPA in the health centres of rural endemic sites or the evaluation centre (EC) of 11 Colombian municipalities and in a reference centre (RC). RESULTS: Samples of 128 patients with suspected CL were included and processed for analysis by RPA vs DE in the EC and RPA vs DE and cPCR in the RC. The RPA performed at the EC was more sensitive (90.4% [95% confidence interval {CI} 81.9 to 95.7]) than the DE (42-67%) and the specificity was 72.7% (95% CI 57.2 to 85.0). Both the sensitivity and specificity increased to 100% when adjusting by the imperfect reference standard analysis method. In the RC, the sensitivity of RPA vs cPCR was 72% and the specificity was 69.8%, while the sensitivity of cPCR vs the DE test was 78.8% and the specificity was 81%. CONCLUSIONS: The higher sensitivity and specificity shown by RPA in the EC, but also its ease and speed of use, justify performing RPA in the health centres of rural endemic sites. In addition, RPA eliminates the subjectivity inherent in the traditional DE.

Asunto(s)

Leishmania , Leishmaniasis Cutánea , Estudios Transversales , Humanos , Leishmania/genética , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/epidemiología , Técnicas de Amplificación de Ácido Nucleico , Estudios Prospectivos , Recombinasas , Sensibilidad y Especificidad

RESUMEN

Single nucleotide polymorphisms (SNPs) have important application value in the research of population genetics, hereditary diseases, tumors, and drug development. Conventional methods for detecting SNPs are typically based on PCR or DNA sequencing, which is time-consuming, costly, and requires complex instrumentation. In this study, we present a duplex probe-directed recombinase amplification (duplex-PDRA) assay that can perform real-time detection of two SNPs (rs6983267 and rs1447295) in four reactions in two tubes at 39°C within 30 min. The sensitivity of duplex-PDRA was 2×103-104 copies per reaction and no cross-reactivity was observed. A total of 382 clinical samples (179 prostate cancer patients and 203 controls) from northern China were collected and tested by duplex-PDRA assay and direct sequencing. The genotyping results were completely identical. In addition, the association analysis of two SNPs with prostate cancer risk and bone metastasis was conducted. We found that the TT genotype of rs6983267 (OR: 0.42; 95%CI: 0.23-0.78; P=0.005) decreased the risk of prostate cancer, while the CA genotype of rs1447295 (OR: 1.89; 95%CI: 1.20-2.96; P=0.005) increased the risk of prostate cancer. However, no association between the two SNPs (rs6983267 and rs1447295) and bone metastasis in prostate cancer was found in this study (P>0.05). In conclusion, the duplex-PDRA assay is an effective method for the simultaneous detection of two SNPs and shows great potential for widespread use in research and clinical settings.

Asunto(s)

Humanos , Masculino , Neoplasias de la Próstata/genética , Cromosomas Humanos Par 8/genética , Análisis Mutacional de ADN/métodos , Polimorfismo de Nucleótido Simple , Estudios de Casos y Controles , China , Predisposición Genética a la Enfermedad , Recombinasas , Genotipo

RESUMEN

Single nucleotide polymorphisms (SNPs) have important application value in the research of population genetics, hereditary diseases, tumors, and drug development. Conventional methods for detecting SNPs are typically based on PCR or DNA sequencing, which is time-consuming, costly, and requires complex instrumentation. In this study, we present a duplex probe-directed recombinase amplification (duplex-PDRA) assay that can perform real-time detection of two SNPs (rs6983267 and rs1447295) in four reactions in two tubes at 39°C within 30 min. The sensitivity of duplex-PDRA was 2×103-104 copies per reaction and no cross-reactivity was observed. A total of 382 clinical samples (179 prostate cancer patients and 203 controls) from northern China were collected and tested by duplex-PDRA assay and direct sequencing. The genotyping results were completely identical. In addition, the association analysis of two SNPs with prostate cancer risk and bone metastasis was conducted. We found that the TT genotype of rs6983267 (OR: 0.42; 95%CI: 0.23-0.78; P=0.005) decreased the risk of prostate cancer, while the CA genotype of rs1447295 (OR: 1.89; 95%CI: 1.20-2.96; P=0.005) increased the risk of prostate cancer. However, no association between the two SNPs (rs6983267 and rs1447295) and bone metastasis in prostate cancer was found in this study (P>0.05). In conclusion, the duplex-PDRA assay is an effective method for the simultaneous detection of two SNPs and shows great potential for widespread use in research and clinical settings.

Asunto(s)

Cromosomas Humanos Par 8/genética , Análisis Mutacional de ADN/métodos , Polimorfismo de Nucleótido Simple , Neoplasias de la Próstata , Estudios de Casos y Controles , China , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Neoplasias de la Próstata/genética , Recombinasas

RESUMEN

Molecular detection of Zika virus (ZIKV) is a key element of outbreak management. Multiple PCR and isothermal ZIKV assays targeting different ZIKV sequences have been published. In this study, we compared a qRT-PCR, 2 RT-LAMP assays (based on different primer design approaches), and an RT-RPA for the detection of African and Asian/American lineages of ZIKV isolates from human, mosquito, and monkey. Results showed that RT-LAMP detected 100% of samples with a time threshold (Tt) of 18.01 ± 11.71 min while qRT-PCR detected 88.88% of samples with a Tt of 58.30 ± 16.58 min and RT-RPA 50% of samples with a Tt of 3.70 ± 0.44 min.

Asunto(s)

Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Infección por el Virus Zika/diagnóstico , Virus Zika/genética , Animales , Brasil , Células Cultivadas , Côte d'Ivoire , Culicidae , Haplorrinos , Humanos , Recombinasas/metabolismo , Reproducibilidad de los Resultados , Senegal , Uganda , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/virología

RESUMEN

ABSTRACT We studied the role of Thermus thermophilus Recombinase A (RecA) in enhancing the PCR signals of DNA viruses such as Hepatitis B virus (HBV). The RecA gene of a thermophilic eubacterial strain, T. thermophilus, was cloned and hyperexpressed in Escherichia coli. The recombinant RecA protein was purified using a single heat treatment step without the use of any chromatography steps, and the purified protein (>95%) was found to be active. The purified RecA could enhance the polymerase chain reaction (PCR) signals of HBV and improve the detection limit of the HBV diagnosis by real time PCR. The yield of recombinant RecA was ∼35 mg/L, the highest yield reported for a recombinant RecA to date. RecA can be successfully employed to enhance detection sensitivity for the diagnosis of DNA viruses such as HBV, and this methodology could be particularly useful for clinical samples with HBV viral loads of less than 10 IU/mL, which is interesting and novel.

Asunto(s)

Proteínas Bacterianas/genética , Virus de la Hepatitis B/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Thermus thermophilus/enzimología , Clonación Molecular , Recombinasas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Expresión Génica , Virus de la Hepatitis B/genética , Reacción en Cadena de la Polimerasa/instrumentación , Thermus thermophilus/genética , Recombinasas/aislamiento & purificación , Recombinasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo

RESUMEN

BACKGROUND: Tools for genome editing in pigs are improving rapidly so that making precise cuts in DNA for the purposes of deleting genes is straightforward. Development of means to replace pig genes with human genes with precision is very desirable for the future development of donor pigs for xenotransplantation. MATERIALS AND METHODS: We used Cas9 to cut pig thrombomodulin (pTHBD) and replace it with a plasmid containing a promoterless antibiotic selection marker and the exon for human thrombomodulin. PhiC31 recombinase was used to remove the antibiotic selection marker to create porcine aortic endothelial cells expressing human instead of pTHBD, driven by the endogenous pig promoter. RESULTS: The promoterless selection cassette permitted efficient enrichment of cells containing correctly inserted transgene. Recombinase treatment of selected cells excised the resistance marker permitting expression of the human transgene by the endogenous pTHBD promoter. Gene regulation was maintained after gene replacement because pig endogenous promoter was kept intact in the correct position. CONCLUSIONS: Cas9 and recombinase technology make orthotopic human for pig gene exchange feasible and pave the way for creation of pigs with human genes that can be expressed in the appropriate tissues preserving gene regulation.

Asunto(s)

Edición Génica/métodos , Porcinos/genética , Trombomodulina/genética , Recolección de Tejidos y Órganos/métodos , Trasplante Heterólogo , Animales , Animales Modificados Genéticamente/genética , Bacteriófagos/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Células Endoteliales , Cultivo Primario de Células , Recombinasas/genética , Transfección/métodos , Proteínas Virales/genética

RESUMEN

We studied the role of Thermus thermophilus Recombinase A (RecA) in enhancing the PCR signals of DNA viruses such as Hepatitis B virus (HBV). The RecA gene of a thermophilic eubacterial strain, T. thermophilus, was cloned and hyperexpressed in Escherichia coli. The recombinant RecA protein was purified using a single heat treatment step without the use of any chromatography steps, and the purified protein (>95%) was found to be active. The purified RecA could enhance the polymerase chain reaction (PCR) signals of HBV and improve the detection limit of the HBV diagnosis by real time PCR. The yield of recombinant RecA was ∼35mg/L, the highest yield reported for a recombinant RecA to date. RecA can be successfully employed to enhance detection sensitivity for the diagnosis of DNA viruses such as HBV, and this methodology could be particularly useful for clinical samples with HBV viral loads of less than 10IU/mL, which is interesting and novel.

Asunto(s)

Proteínas Bacterianas/genética , Clonación Molecular , Virus de la Hepatitis B/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Recombinasas/genética , Thermus thermophilus/enzimología , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Virus de la Hepatitis B/genética , Reacción en Cadena de la Polimerasa/instrumentación , Recombinasas/aislamiento & purificación , Recombinasas/metabolismo , Thermus thermophilus/genética

RESUMEN

We investigated the epidemiology and clonality of 175 nonrepetitive methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical specimens collected between 2011 and 2012 in Kinta Valley in Malaysia. Molecular tools such as polymerase chain reaction, pulsed-field gel electrophoresis, and staphylococcal protein A (spa) typing were used. Our study revealed the predominance of three closely related ermA+ SCCmec type III pulsotypes belonging to spa type t037 (Brazilian-Hungarian clone), which were deficient in the locus F, but positive for the ccrC gene in majority (65.7%) of the MRSA infections in this region. The first evidence of SCCmec type II MRSA in the country, belonging to spa type t2460, was also noted. Although the carriage of pvl gene was uncommon (8.6%) and mostly confined to either SCCmec type IV or SCCmec type V isolates, most of these isolates belonged to spa types t345 or t657, which are associated with the Bengal-Bay CA-MRSA clone. Interestingly, spa t304 and t690 SCCmec type IV pvl+ were also detected among the MRSA isolates. Data from this study show the rise of uncommon clones among MRSA isolates in Malaysia.

Asunto(s)

Proteínas Bacterianas/genética , Genes Bacterianos/genética , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Recombinasas/genética , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Proteína Estafilocócica A/genética , Brasil , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Humanos , Malasia/epidemiología , Epidemiología Molecular/métodos

RESUMEN

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance.

Asunto(s)

Heces/parasitología , Giardia lamblia/aislamiento & purificación , Giardiasis/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Recombinasas/metabolismo , Giardiasis/epidemiología , Humanos , Perú/epidemiología , Recombinasas/química , Sensibilidad y Especificidad

RESUMEN

The development of meiotic division and associated genetic recombination paved the way for evolutionary changes. However, the secondary and tertiary structure and functional domains of many of the proteins involved in genetic recombination have not been studied in detail. We used the human Dmc1 gene product along with secondary and tertiary domain structures of Escherichia coli RecA protein to help determine the molecular structure and function of maize Dmc1, which is required for synaptonemal complex formation and cell cycle progression. The maize recombinase Dmc1 gene was cloned and characterized, using rice Dmc1 cDNA as an orthologue. The deduced amino acid sequence was used for elaborating its 3-D structure, and functional analysis was made with the CDD software, showing significant identity of the Dmc1 gene product in Zea mays with that of Homo sapiens. Based on these results, the domains and motives of WalkerA and WalkerB as ATP binding sites, a multimer site (BRC) interface, the putative ssDNA binding L1 and L2 loops, the putative dsDNA binding helix-hairpin-helix, a polymerization motif, the subunit rotation motif, and a small N-terminal domain were proposed for maize recombinase Dmc1.

Asunto(s)

Proteínas de Ciclo Celular/genética , Genoma de Planta , Meiosis , Proteínas de Plantas/genética , Recombinasas/genética , Zea mays/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Filogenia , Conformación Proteica , Alineación de Secuencia , Zea mays/genética

RESUMEN

VIPER was initially characterized as a 2326bp LTR-like retroelement associated to SIRE, a short interspersed repetitive element specific of Trypanosoma cruzi. It carried a single ORF that coded for a putative reverse transcriptase-RNAse H protein, suggesting that it could be a truncated copy of a longer retroelement. Herein we report the identification and characterization of a complete 4480bp long VIPER in the T. cruzi genome. The complete VIPER harbored three non-overlapped domains encoding for a GAG-like, a tyrosine recombinase and a reverse transcriptase-RNAse H proteins. VIPER elements were also found in the genomes of Trypanosoma brucei and Trypanosoma vivax, but not in Leishmania sp. On the basis of its reverse transcriptase phylogeny, VIPER was classified as an LTR retroelement. However, VIPER was structurally related to the tyrosine recombinase encoding retroelements, DIRS and Ngaro. Phylogenetic analysis showed that VIPER's tyrosine recombinase grouped with the transposases RCI1 of Escherichia coli and Ye24 and Ye72 of Haemophilus influenzae within a major branch of prokaryotic recombinases. Taken together, VIPER's structure, the nature of its tyrosine recombinase, the unique features of its reverse transcriptase catalytic consensus motif and the fact that it was found in Trypanosomes, an early branching eukaryote, suggest that VIPER may be the closest relative of the founder element of the tyrosine recombinase encoding retrotransposons known up to date. Our analysis revealed that tyrosine recombinase-encoding retroelements were originated as early in evolution as non-LTR retroelements and suggests that VIPER, Ngaro and DIRS elements may constitute a third group of retrotransposons, distinct from both LTR and non-LTR retroelements.

Asunto(s)

Genoma de Protozoos , Recombinasas/genética , Retroelementos/genética , Trypanosoma cruzi/genética , Secuencia de Aminoácidos , Animales , Biología Computacional , ADN Nucleotidiltransferasas/genética , Productos del Gen gag/genética , Haemophilus influenzae/genética , Leishmania/genética , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , ADN Polimerasa Dirigida por ARN/genética , Ribonucleasa H/genética , Homología de Secuencia , Trypanosoma brucei brucei/genética , Trypanosoma vivax/genética