Amplification and sequencing of genomic DNA fragments encoding cysteine proteases from protozoan parasites

Cysteine protease gene fragments from three protozoan parasites Trypanosoma cruzi, Trypanosoma brucei, and Entamoeba histolytica were amplified by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotide primers. The primers used for the amplification were designed based upon amino acid sequences flanking the active site cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteases analyzed to date. The amplified DNA fragments, representing approximately 70% of the coding regions of the cysteine protease genes, were subcloned and sequenced. Sequence analysis and alignment showed significant sequence similarity to other members of the eukaryotic cysteine protease family (45% identical to chicken cathepsin L) and conservation of the cysteine, histidine, and asparagine residues which form the catalytic triad. These gene fragments provide molecular probes for further analysis of the structure and function of these important metabolic enzymes.     

RAPD–SCAR marker and genetic relationship analysis of three Demodex species (Acari: Demodicidae)

For a long time, classification of Demodex mites has been mainly based on their hosts and phenotype characteristics. The study was the first to conduct molecular identification and genetic relationship analysis for six isolates of three Demodex species by random amplified polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) marker. Totally, 239 DNA fragments were amplified from six Demodex isolates with 10 random primers in RAPD, of which 165 were polymorphic. Using a single primer, at least five fragments and at most 40 in the six isolates were amplified, whereas within a single isolate, a range of 35-49 fragments were amplified. DNA fingerprints of primers CZ 1-9 revealed intra- and interspecies difference in six Demodex isolates, whereas primer CZ 10 only revealed interspecies difference. The genetic distance and dendrogram showed the intraspecific genetic distances were closer than the interspecific genetic distances. The interspecific genetic distances of Demodex folliculorum and Demodex canis (0.7931-0.8140) were shorter than that of Demodex brevis and D. canis (0.8182-0.8987). The RAPD-SCAR marker displayed primer CZ 10 could be applied to identify the three Demodex species. The 479-bp fragment was specific for D. brevis, and the 261-bp fragment was specific for D. canis. The conclusion was that the RAPD-SCAR multi-marker was effective in molecular identification of three Demodex species. The genetic relationship between D. folliculorum and D. canis was nearer than that between D. folliculorum and D. brevis.     

Development of a strain-specific SCAR marker for the detection of Trichoderma atroviride 11, a biological control agent against soilborne fungal plant pathogens

The genus Trichoderma includes biocontrol agents (BCAs) effective against soilborne plant pathogenic fungi. Several potentially useful strains for biological control are difficult to distinguish from other strains of Trichoderma found in the field. So, there is a need to find ways to monitor these strains when applied to natural pathosystems. We have used random amplified polymorphic DNA (RAPD) markers to estimate genetic variation among sixteen strains of the species T. asperellum, T. atroviride, T. harzianum, T. inhamatum and T. longibrachiatum previously selected as BCAs, and to obtain fingerprinting patterns. Analysis of these polymorphisms revealed four distinct groups, in agreement with previous studies. Some of the RAPD products generated were used to design specific primers. Diagnostic PCR performed using these primers specifically identify the strain T. atroviride 11, showing that DNA markers may be successfully used for identification purposes. This SCAR (sequence-characterised amplified region) marker can clearly distinguish strain 11 from other closely related Trichoderma strains. Received: 9 June 2000 / Accepted: 5 October 2000     

特异性引物PCR鉴定鸡球虫种类与卵囊纯度的研究

为了建立鸡球虫种类的快速分子生物学鉴定方法,检测实验室保存虫株受污染状况,分别对单卵囊分离繁殖及实验室长期传代保存的6株巨型艾美耳球虫（EMSH01、EM4101、EMES01、EMBA01、EMTY01和EMTO01）、4株柔嫩艾美耳球虫（ETDS01、ETGD01、ETAD和ETAM）、1株堆形艾美耳球虫（EA1201）、1株变位艾美耳球虫（EMIS01）、1株毒害艾美耳球虫（ENGD01）收集卵囊,纯化、提取总DNA,根据巨型艾美耳球虫、柔嫩艾美耳球虫、堆形艾美耳球虫的RAPD和SCAR分子标记、ITS-1区序列分别设计Tn-F与Tn-R、Mx-F与Mx-R、Ac-F与Ac-R、ET-1与ET-2、EM-1与EM-2、EA-1与EA-2等6对特异性引物,对13个虫株分别进行PCR扩增,1%琼脂糖电泳分析片段大小.结果显示：用引物Tn-F与Tn-R、ET-1与ET-2对ETDS01、ETGD01、ETAD、ETAM、EMTO01扩增出特异性条带,其余4种8个虫株未见条带;用引物Mx-F与Mx-R、EM-1与EM-2对EMSH01、EM4101、EMES01、EMBA01、EMTY01和EMTO01扩增出特异性条带,其余4种7个虫株未见条带;用引物Ac-F与Ac-R、EA-1与EA-2对EA1201扩增出特异性条带,其余4种12个虫株未见条带.结果说明特异性引物PCR方法鉴定的5种球虫与原常规生物学方法鉴定的结果一致,检测出保存的巨型艾美耳球虫EMTO01虫株受柔嫩艾美耳球虫污染,其余虫株未发现交叉污染.研究证明利用特异性引物建立的PCR方法可用于鸡球虫种类与卵囊纯度的鉴定.     

Schizodeme analysis of Trypanosoma cruzi stocks from South and Central America by analysis of PCR-amplified minicircle variable region sequences

Kinetoplast DNA (kDNA) was isolated from 56 stocks of Trypanosoma cruzi isolated from human patients, animals and insects from Brazil, Venezuela, Colombia and Costa Rica. Comparison of the patterns of digested kDNA on acrylamide gels led to the grouping of several stocks into two schizodemes. Schizodeme analysis was also performed using a set of 330-bp fragments representing all the variable regions of the minicircle DNA molecules, which were obtained by PCR amplification of the kDNA using conserved region primers. The results of this analysis were consistent with the analysis using total kDNA, but the more informative restriction profiles allowed the construction of additional schizodemes. In addition, two oligomers were generated from variable region sequences of cloned minicircles from a Y and a Cl strain, and these were used as schizodeme-specific probes to detect homologous sequences in the amplified minicircle DNAs. The results indicate that a combination of restriction enzyme fingerprinting and hybridization of amplified variable region minicircle DNA with schizodeme-specific probes can be used for both sensitive detection and classification of T. cruzi.     

Molecular genetic characterization of different Trypanosoma cruzi strains and comparison of their development in Mus musculus and Calomys callosus

Trypanosoma cruzi populations are characterized by diverse morphology, heterogeneous biological behavior, high genetic variability, and distinctly different clinical courses. The first objective of this work was to characterize different strains of T. cruzi with various molecular markers [simple-sequence-repeat PCR, randomly amplified polymorphic DNA (RAPD)-PCR, mini-exon genes]. All examined strains could be divided into two major lineages. Only one strain showed a different banding pattern in RAPD-PCR, which could be a further indication of the existence of a third lineage. The second aim was to examine the biological behavior of the different strains. Two animal models, Calomys callosus and Mus musculus, were infected. The results provide strong evidence that the biological behavior of the strains is not only lineage-specific. It appears that all factors, such as the infecting strain belonging to a certain lineage, the predominant morphological form of the isolate, and the immune response of the respective infected host, play an important role in the course of this infection.     

Random amplified polymorphic DNA of mosquito species and populations (Diptera: Culicidae): techniques, statistical analysis, and applications.

A polymerase chain reaction (PCR)-based method is described for the identification and differentiation of mosquito species and populations. The method, described first by Williams et al. (1990), employs single 10 base-long primers of arbitrary DNA sequence and results in the amplification of random segments of DNA known as random amplified polymorphic DNA (RAPD). We wished to determine if RAPD of mosquito DNA could be used for the differentiation of species and populations, identification of unknown specimens, and the reconstruction of phylogeny. RAPD of mosquito DNA results in the amplification of a series of DNA fragments of varying length. Most amplified fragments are unique to an individual; however, our data indicated that in each of the five species of Aedes examined, some fragments are species-specific and are present in all individuals of that species. This enabled us to derive a diagnostic profile for each of the five species. A nearest-neighbor analysis of all the amplified DNA fragments discriminated among species on a multivariate basis. Several individuals of Aedes albopictus (Skuse), included in the analysis as "unknowns," were correctly identified as belonging to Ae. albopictus. UPGMA clustering of presence-absence data enabled the separation of different Aedes species as well as different populations of Ae. albopictus. The entomological applications of RAPD include the construction of diagnostic profiles for species identification and differentiation among conspecific populations.     

Toxoplasma gondii virulence markers identified by random amplified polymorphic DNA polymerase chain reaction

Genomic DNAs from 35 Toxoplasma gondii strains were amplified by random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) using 18 arbitrary 10-mer primers. At least four primers were found to generate DNA fragments that discriminate the 35 T. gondii strains into a genotype of virulent strains and a genotype of avirulent strains. Primer B12 was found to generate a virulence-specific fragment and primers B5, C8, and C20 were found to generate avirulence-specific fragments, which in all cases clearly identified either the virulence phenotype or the avirulence phenotype, respectively. In addition, the DNA polymorphic bands detected were analyzed by parsimony and distance analysis. A similar genetic relationship among the T. gondii strains was determined by the two phylogenetic methods, which use completely different assumptions. Consistent with the division of the 35 strains into a genotype of virulent strains and a genotype of avirulent strains, both analyses revealed 2 clonal lineages directly correlated with murine virulence. These results strongly support the hypothesis that the genus Toxoplasma may actually contain two clonal lineages correlated with virulence, which have evolved independently following their initial separation. Received: 1 October 1996 / Accepted: 15 October 1996     

Polymerase chain reaction amplification of Trypanosoma cruzi kinetoplast minicircle DNA isolated from whole blood lysates: diagnosis of chronic Chagas' disease.

A 6 M guanidine-HCl/0.2 M EDTA solution was used to lyse and store whole blood specimens. DNA stored in guanidine-EDTA-blood (GEB) lysate was found to be undegraded after incubation at 37 degrees C for 1 month, suggesting that this represents an appropriate reagent for transport of blood samples from the field to a laboratory for analysis. Trypanosoma cruzi kinetoplast DNA in GEB lysate can be cleaved using the chemical nuclease, 1,10-phenanthroline-copper ion (OP-Cu2+). This procedure liberates linearized minicircle molecules from network catenation, distributing them throughout the lysate, and allowing a small aliquot of the original lysate to be analyzed by PCR amplification. This increases the sensitivity of the method dramatically for the detection of small numbers of trypanosomes in a large volume of blood. DNAs isolated from aliquots of T. cruzi-positive GEB lysates were polymerase chain reaction (PCR)-amplified with 3 sets of T. cruzi-specific kDNA minicircle primers, yielding the 83-bp and 122-bp conserved region fragments and the 330-bp variable region fragments. The PCR products were analyzed by gel electrophoresis and/or hybridization. Results indicate that a single T. cruzi cell in 20 ml of blood can be detected by this method. Blood samples from several chronic chagasic patients were tested. Amplification of T. cruzi kDNA minicircle sequences was obtained in al cases, even when xenodiagnosis was negative. This PCR-based test should prove useful as a replacement or complement for xenodiagnosis or serology in clinical and epidemiological studies of chronic Chagas' disease.     

Genetic polymorphism in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> I isolated from Brazilian Northeast triatomines revealed by low-stringency single specific primer–polymerase chain reaction

Different molecular markers have been employed for typing Trypanosoma cruzi strains from endemic areas of Chagas disease. The low-stringency single specific primer-polymerase chain reaction (LSSP-PCR) has been a sensitive and informative technique that uses the variable region of kinetoplast DNA minicircles as a genetic marker, allowing detection of DNA sequence variation. In the present study, we analyzed the intra-lineage genetic variability of the T. cruzi strains obtained from triatomine feces collected on filter paper FTA card by LSSP-PCR. The hybridization of the PCR products with a probe for the subgenus Schizotrypanum and a clone-specific probe from Dm28c confirmed the subgenus as T. (S.) cruzi and respective lineages as T. cruzi I. Phenetic analysis showed the presence of three clusters that diverged by different coefficients of similarity. Thirteen T. cruzi I genotypes were observed circulating among Triatoma pseudomaculata and Rhodnius nasutus from peridomiciliary and natural environments in five peri-urban and urban localities of Jaguaruana, Ceará, Brazil. These data indicate the importance of the circulation of T. cruzi I genotypes among T. pseudomaculata and R. nasutus in different environments and the possible risk of Chagas disease domestic transmission.     

Eimeria parasites of domestic fowl: genetic relationships of different isolates estimated from random amplified polymorphic DNA

Genetic relationships between Eimeria parasites of domestic fowl, including three isolates of E. acervulina, one of E. maxima, four of E. mitis, six of E. praecox, one of E. tenella, and two of uncertain identity, were analyzed by random amplified polymorphic DNA (RAPD) and the unweighted pair-group method with arithmetic mean, using 12 primers. Each primer amplified 4-34 DNA fragments/isolate. The similarity coefficients and phenograms were calculated from RAPD products with 230-2,000 bp in silver-stained polyacrylamide gels. Some primers generated RAPD markers which were species-specific for E. acervulina, E. mitis, and E. praecox. The phenograms revealed six clusters, each corresponding to an individual Eimeria species. The pBP and pBP2 isolates, of uncertain identity, clustered with the E. acervulina isolates (100% bootstrap). The intraspecific relationships showed certain degree of genetic isolation of the Eimeria populations, and it was associated with broiler house and with geographic separation.     

Predominance of Trypanosoma cruzi lineage I in Mexico

Randomly amplified polymorphic DNA (RAPD) has emerged as an effective genetic marker for analysis of Trypanosoma cruzi population variability. This method has been used to study the genetic variability of Mexican T. cruzi stocks and to relate these results to previous classifications. High clonal diversity was observed among the Mexican populations: 24 RAPD types were scored among 56 stocks analyzed. Only two stocks (3.6%) belonged to the T. cruzi II lineage, while all others belonged to T. cruzi I. The robustness of these clusters was statistically highly significant. Mexican T. cruzi I stocks formed a homogeneous group with reduced genetic distances among its members. Parasites from this group were isolated from both domestic and sylvatic cycles over a broad geographic area in Mexico. The two Mexican stocks classified as T. cruzi II (isolated from sylvatic cycles) were of the same RAPD type, although they were not closely related to the three reference T. cruzi II stocks circulating in domestic cycles in Argentina, Brazil, Bolivia, and Chile. These stocks were also unrelated to the formerly named Zymodeme III.     

Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.     

Detection of Trypanosoma cruzi in blood specimens of chronic chagasic patients by polymerase chain reaction amplification of kinetoplast minicircle DNA: comparison with serology and xenodiagnosis.

A panel of 114 blood samples from chronic chagasic patients and nonchagasic patients was screened for Trypanosoma cruzi by xenodiagnostic, serologic, and polymerase chain reaction (PCR) amplification tests. Blood samples were preserved in a guanidine-EDTA buffer, and total blood DNA was isolated after chemical nuclease cleavage with 1,10-phenanthroline-copper ion and used as a template for PCR amplification of the conserved and variable regions of T. cruzi minicircle molecules. The PCR products were screened by Southern blot hybridization with a digoxigenin-labeled oligonucleotide probe specific for the conserved region of the minicircle. The method showed a sensitivity of 100% compared with the serologic test. In addition, all of the serology-positive, xenodiagnosis-negative samples were positive by PCR. This demonstrates that PCR amplification of T. cruzi kinetoplast minicircle DNA could replace xenodiagnosis for evaluation of parasitemia in chronic chagasic patients and could serve as a complement for serologic testing in the screening of blood bank donors.     

Individual identification by DNA polymorphism using formalin-fixed placenta with whole genome amplification

Polymerase chain reaction (PCR) amplification using formalin-fixed material is very limited. In the present study the use of 6 week formalin-fixed placenta for individual identification was examined based on DNA analyses. The objective of the examination was to prove whether the placenta was from a woman who had just given birth. DNA extraction was carried out from the maternal blood sample and from the formalin-fixed placental samples composed of three parts: maternal side, infant side and umbilical cord. One minisatellite (D1S80), 12 short tandem repeat (STR) polymorphisms and amelogenin X, Y were investigated. All the polymorphic systems were detected in the maternal blood sample. The majority of the DNA isolated from the placental tissues had molecular weights of approximately 500 bp, and only two to four STR loci were amplified using the DNA. In order to amplify more DNA polymorphic markers from the formalin-fixed tissues, whole genome amplification was performed. After amplification by degenerate oligonucleotide-primed PCR (DOP-PCR), the products contained DNA with increased molecular weight up to >10 kbp. More DNA loci were typed using the DOP-PCR products. Furthermore, large molecular size fragments were purified from the DOP-PCR products by agarose electrophoresis, and then the D1S80 locus and 12 STR loci were successfully amplified using these fragments.     

Development of specific sequence-characterized amplified region markers for detecting Histoplasma capsulatum in clinical and environmental samples

Sequence-characterized amplified region (SCAR) markers, generated by randomly amplified polymorphic DNA (RAPD)-PCR, were developed to detect Histoplasma capsulatum selectively in clinical and environmental samples. A 1,200-bp RAPD-PCR-specific band produced with the 1281-1283 primers was cloned, sequenced, and used to design two SCAR markers, 1281-1283(220) and 1281-1283(230). The specificity of these markers was confirmed by Southern hybridization. To evaluate the relevance of the SCAR markers for the diagnosis of histoplasmosis, another molecular marker (M antigen probe) was used for comparison. To validate 1281-1283(220) and 1281-1283(230) as new tools for the identification of H. capsulatum, the specificity and sensitivity of these markers were assessed for the detection of the pathogen in 36 clinical (17 humans, as well as 9 experimentally and 10 naturally infected nonhuman mammals) and 20 environmental (10 contaminated soil and 10 guano) samples. Although the two SCAR markers and the M antigen probe identified H. capsulatum isolates from different geographic origins in America, the 1281-1283(220) SCAR marker was the most specific and detected the pathogen in all samples tested. In contrast, the 1281-1283(230) SCAR marker and the M antigen probe also amplified DNA from Aspergillus niger and Cryptococcus neoformans, respectively. Both SCAR markers detected as little as 0.001 ng of H. capsulatum DNA, while the M antigen probe detected 0.5 ng of fungal DNA. The SCAR markers revealed the fungal presence better than the M antigen probe in contaminated soil and guano samples. Based on our results, the 1281-1283(220) marker can be used to detect and identify H. capsulatum in samples from different sources.     

PCR技术在单克隆抗体cDNA的筛选及标记同位素探针上的应用

以往对cDNA克隆鉴定过程中,经斑点杂交筛选出的克隆数较多,在进一步的鉴定中不可避免地要进行质粒扩增、抽提、纯化酶解、片段分离等许多步骤。本文对这一步的鉴定采用了PCR技术,这样便可省去酶解及片段分离等繁琐步骤。且由于扩增片段纯净,故可直接用于Southern blot鉴定及DNA序列分析。 本文另一新的探索是利用PCR法标记探针,尤其是小片段DNA探针,其不同于常用的缺口平移和随机引物延伸标记。由于它在扩增过程中只有位于两个引物间的片段才能标上同位素,所以标记后的探针量大、比放射性强,加之PCR合成仪的出现,使此法更加高效、简便安全。     

The evolution of two Trypanosoma cruzi subgroups inferred from rRNA genes can be correlated with the interchange of American mammalian faunas in the Cenozoic and has implications to pathogenicity and host specificity

The agent of Chagas disease, Trypanosoma cruzi, is divided into two highly divergent genetic subgroups, lineages 1 and 2, which include all typed strains isolated from humans, insect vectors, and sylvatic mammals. The evolutionary origin of these two T. cruzi lineages and the clinical importance of their identification, have been the subject of intense debate. Here, using molecular phylogenetic analysis, we found that the distance between the two T. cruzi lineages is equivalent to the distance between genera Leishmania and Endotrypanum. Also, we confirmed that T. rangeli is more closely related to T. cruzi than to T. brucei using the rDNA sequence from a human strain of T. rangeli. Phylogenetic trees based on small subunit rDNA sequences further suggest that the two T. cruzi lineages diverged between 88 and 37 million years (Myr) ago. We hypothesize that lineage 2 is indigenous to South America while lineage 1 has been introduced to South America recently, along with North American placental mammals, after the connection of the Americas in the Pliocene (5 Myr ago) or with caviomorph rodents and primates in the Oligocene (38 Myr ago). This would explain the preferential association of T. cruzi lineage 2 with marsupials and of lineage 1 with human disease. These two T. cruzi lineages are likely to be distinct species, or at least subspecies, because of their different ecological and epidemiological traits and estimated long period of independent evolution.     

Genetic heterogeneity in the Trypanosoma brucei gambiense genome analysed by random amplification of polymorphic DNA

 The random amplification of polymorphic DNA (RAPD) technique has the potential to produce large amounts of characterisation data very quickly and simply, using far less DNA than conventional restriction-fragment-length polymorphism (RFLP) analysis. In the present study we assessed genetic heterogeneity among 34 Trypanosoma brucei gambiense isolates from various endemic areas in Africa by the RAPD technique using 8 arbitrary primers and compared the results with those obtained previously from RFLP analysis of polymorphisms in 5 variant surface glycoprotein (VSG) genes. The isolates were compared both among themselves and with 3 T. b. non-gambiense isolates. Most of the primers produced RAPD profiles specific for T. b. gambiense, with 4 primers showing marked polymorphisms between T. b. gambiense and non-gambiense stocks. These primers also showed minor variations between the T. b. gambiense stocks, and 2 revealed differences between Cameroonian stocks. These results were comparable with those produced by RFLP analysis, where certain polymorphisms are characteristic of T. b. gambiense. Numerical analysis showed a high correlation between the RAPD and RFLP data, with genetic variation being detected at a finer level by RAPD analysis. We conclude that RAPD analysis provides a simple and accurate method for the characterisation of T. b. gambiense. Received: 15 February 1996 / Accepted: 15 March 1996     

Use of random amplified polymorphic DNA for identification of ruminant trichostrongylid nematodes

The aim of this work was to evaluate random amplified polymorphic DNA (RAPD) as a source of markers for species identification and phylogenetic analysis of ruminant trichostrongylid nematodes. As these nematodes are often polymorphic, species identification may be difficult. We tested eight species and several of their morphs:Haemonchus contortus (three vulvar morphotypes: flap, smooth, and knobbed),Teladorsagia circumcincta, Ashworthius gagarini, Spiculopteragia boehmi, Ostertagia leptospicularis (and its morphOstertagia kolchida), Cooperia oncophora (and its morphC. surnabada), Trichostrongylus colubriformis, andT. vitrinus. With five chosen 10-mer primers, genetic variations were assessed among individuals of each species or morphotype. In trichostrongylid nematodes, the identification of species is possible by means of RAPD on adult or larva DNA extracts, although the variability observed within species was very important for most species studied. The use of RAPD in phylogenetics studies is conversely questionable for this superfamily of parasitic nematodes. The interspecific distances were always larger than the intraspecific ones and did not vary much (between 0.8 and 0.9); they would not be of much use in the construction of a phylogenetic tree, at least for the species and the primers involved in this study.