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WO1997030176A1 - Detection a usage diagnostique de plasmodium falciparum - Google Patents

Detection a usage diagnostique de plasmodium falciparum Download PDF

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Publication number
WO1997030176A1
WO1997030176A1 PCT/AU1997/000076 AU9700076W WO9730176A1 WO 1997030176 A1 WO1997030176 A1 WO 1997030176A1 AU 9700076 W AU9700076 W AU 9700076W WO 9730176 A1 WO9730176 A1 WO 9730176A1
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WIPO (PCT)
Prior art keywords
pcr
primers
falciparum
seq
biological sample
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Ceased
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PCT/AU1997/000076
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English (en)
Inventor
Allan James Saul
Qin Cheng
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QIMR Berghofer Medical Research Institute
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Queensland Institute of Medical Research QIMR
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Priority to AU15856/97A priority Critical patent/AU1585697A/en
Publication of WO1997030176A1 publication Critical patent/WO1997030176A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to a diagnostic assay procedure and more particularly to an assay procedure for Plasmodium falciparum malaria parasite.
  • the present invention also provides a diagnostic kit for detecting P. falciparum.
  • the present invention permits for the first time a highly sensitive and quantitative assay for the malaria parasite and greatly facilitates diagnostic and therapeutic mitums for this debilitating disease.
  • the assay of the present invention is particularly useful for monitoring and screening the efficacy of a malarial vaccine.
  • the detection and enumeration of malaria parasites in the blood is essential for managing the treatment of individuals infected with malaria and for epidemiological surveys required for malaria control programs.
  • Microscopic examination is inexpensive and does not require exotic or labile reagents. However, its efficiency is highly dependent on the skill of the trained microscopist. Under ideal conditions, with a proficient operator, microscopic examination has a lower limit of detection of about 20 parasites per microlitre. These limits can only be achieved with a highly trained and proficient microscopist with an extended period of examination. For example, in local practice, two microscopists examine a thick film for 15 minutes each before scoring it as parasite negative.
  • a test needs a routine detection level at least 10 fold greater than microscopy (i.e. a detection threshold of ⁇ 1 parasite per microlitre).
  • a detection threshold of ⁇ 1 parasite per microlitre.
  • people returning from endemic areas, who get sick will take anti-malarial drugs prior to clinical examination.
  • a malaria test needs to be much more sensitive since it needs to be able to detect low frequencies of dead or dying parasites (i.e. a detection limit of ⁇ 0.1 parasite per microlitre).
  • tests other than microcopy have been proposed (3-11). These include revised microscopic tests such as the QBC test from Becton Dickinson and a number of Dip-stick tests for rapid non-microscopic diagnosis. However, these have a detection limit similar to or only marginally greater than conventional microscopy. Tests based on DNA detection and amplification have also been proposed, but none have been commercially developed. Although several of these are able to achieve a high sensitivity, complex and time consuming detection procedures make them difficult and expensive to use in a routine pathology laboratory and take too long to produce a result able to influence the course of treatment.
  • a new quantitative polymerase chain reaction based test for malaria is provided.
  • This test is readily able to detect a single parasite and the sensitivity is limited only by sampling considerations. It is 1000 fold more sensitive than microscopy, detection is rapid and the assay inexpensive.
  • the inclusion of an internal control allows positive verification that the test is valid and a comparison of the relative intensity of the internal standard and the test bands gives a measure of the parasite density.
  • the assay of the present invention is particularly useful in determining the efficacy of a malaria vaccine thus reducing the need for expensive field trials based on natural challenge.
  • one aspect of the present invention contemplates a method for detecting Plasmodium falciparum in a biological sample, wherein said method comprises isolating DNA from said biological sample wherein said DNA would include P. falciparum DNA if present in said biological sample and subjecting said isolated DNA to polymerase chain reaction (PCR) using at least one set of primers from a conserved sequence of a multicopy DNA family in the P. falciparum genome and then detecting the product of said PCR wherein the presence of said PCR product in indicative of the presence of P. falciparum.
  • PCR polymerase chain reaction
  • the conserved sequence is the subtelemeric variable open reading frame which is abbreviated herein to "STEVOR".
  • STOR subtelemeric variable open reading frame
  • this family of sequences is polymorphic, short conserved regions have been detected which permit PCR primers to be identified.
  • the use of this multicopy family gives a substantial increase in sensitivity compared with amplification of single copy genes.
  • the primers are oligonucleotides selected from the list comprising or consisting of SEQ LD NO:l to SEQ LD NO:6, inclusive. These primers are identified herein in Table 1. However, the present invention extends to other suitable sequences within STEVOR.
  • a method for detecting P. falciparum in a biological sample comprising isolating DNA from said biological sample wherein said DNA would include P. falciparum DNA if present in said biological sample and subjecting said isolated DNA to at least one round of PCR using sets of primers capable of amplifying STEVOR sequences on P. falciparum and then detecting the presence of the PCR product wherein the presence of said product is indicative of the presence of P. falciparum.
  • the primers are selected from the list comprising or consisting of SEQ LD NO: 1 to SEQ LD NO:6, inclusive.
  • a first round may be conducted using a first set of primers selected from SEQ LD NO: 1 to SEQ LD NO:6 and the second or subsequent round(s) conducted using a set of primers also selected from SEQ LD NO: 1 to SEQ LD NO:6.
  • Both sets of primers may be the same or different and it is particularly preferred that the second set of primers be nested between the first set of primers.
  • a method for detecting P. falciparum in a biological sample comprising isolating DNA from said biological sample wherein said DNA would include P. falciparum DNA if present in said biological sample and subjecting said isolated DNA to a first round PCR using a set of primers capable of amplifying STEVOR sequences on P. falciparum and then subjecting the resulting PCR product to a second round PCR wherein at least one of the PCR primers used in the second round PCR is nested between the primers used in the first round PCR and then detecting the presence of the second round PCR product wherein the second round PCR product is indicative of P. falciparum.
  • the primers are selected from SEQ LD NO: 1 to SEQ LD NO:6.
  • the biological sample used in accordance with the present invention comprises any source of blood cells potentially parasitized with P. falciparum.
  • whole blood is used from a human subject.
  • an assayable amount of blood is taken such as from about 10 ⁇ l to about 50 ml, preferably from about 20 ⁇ l to about 20 ml, more preferably from about 100 ⁇ l to about 10 ml and most preferably around about 100 ⁇ l.
  • the red blood cells are lysed and the total DNA extracted in an appropriate volume of water. Conveniently, approximately 1 ml of water is used.
  • Such a solution contains a very high concentration and may require the addition of higher taq polymerase and magnesium ion concentration. In any event under these conditions, a detection limit is achieved of about 10 parasites per ml or about 1000 fold greater sensitivity than conventional microscopy.
  • PCR is the preferred detection means
  • the present invention extends to any nucleic acid-based detection means such as nucleic acid hybridisation techniques.
  • the invention further encompasses the use of different assay formats of nucleic acid-based detection means, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (LRS-PCR), inverse polymerase reaction (iPCR) and reverse transcription polymerase chain reaction (RT-PCR), amongst others.
  • RFLP restriction fragment length polymorphism
  • AFLP amplified fragment length polymorphism
  • SSCP single-strand chain polymorphism
  • ATD amplification and mismatch detection
  • LRS-PCR interspersed repetitive sequence polymerase chain reaction
  • iPCR inverse polymerase reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • the Plasmodium target nucleic acid may be labelled with a reporter molecule capable of producing an identifiable signal (e.g. a radioisotope such as 3 P or 3S S or a biotinylated molecule).
  • a reporter molecule capable of producing an identifiable signal (e.g. a radioisotope such as 3 P or 3S S or a biotinylated molecule).
  • the present invention extends to all lines, sub-species and varieties of P. falciparum and may be modified to permit distinction between variants of the malaria parasite.
  • the assay protocol of the present invention may also include an internal control to facilitate measuring the relative amount of parasite DNA in the blood sample.
  • plasmid DNA carrying the STEVOR sequence may be amplified and its concentration determined using any convenient means, such as UV spectroscopy. Conveniently the plasmid DNA control is added to the blood before extraction of total DNA.
  • the present invention further contemplates a kit for detecting P. falciparum in a biological sample, said kit comprising in compartmentalised form a first container adapted to contain a set of primers based on a STEVOR sequence, optionally a second container adapted to contain a second set of primers based on a STEVOR sequence wherein at least one primer in the second set is nested between the primers of the first set, a third container adapted to contain reagents for PCR and optionally a fourth container to receive the biological sample.
  • Other containers may be included in order to facilitate the PCR.
  • the kit may also include electrophoretic apparatus, detecting reagents and instructions for use. Another aspect of the present invention is directed to the use of primers based on the STEVOR sequence in a PCR to detect P. falciparum DNA in a biological sample.
  • Still a further aspect of the present invention contemplates an agent comprising one or more sets of primers based on the STEVOR sequence for use in a PCR to detect P. falciparum DNA in a biological sample.
  • Figure 1 is a photographic representation showing amplification of the STEVOR sequence from 13 laboratory lines of P. falciparum.
  • Figure 2 is a photographic representation showing a comparison of the amplification of the STEVOR sequence with a single copy gene (MSA-1 pgl9).
  • PCR reaction mixtures contained: 1) 200pg; 2) 20pg; 3) 2pg; 4) 0.2pg; 5) 0.02pg; 6) 0.002pg; 7) 0.0002pg; and 8) Opg of total P. falciparum DNA, respectively.
  • Figure 3 is a photographic representation showing standards for the detection of malaria parasites in blood.
  • DNA in 3ml blood samples containing 300, 100, 30, 10 or 0 parasitised red cells per ml was extracted and amplified as detailed in Example 2.
  • the resulting amplified DNA after separation by gel electrophoresis gives a 188bp product derived from the parasite DNA and a 133bp product from the internal control.
  • the total amount of DNA amplified is approximately constant, hence, the ratio of parasite to control band is an indicator of the number of parasites present in the sample.
  • Figure 4 is a photographic representation of amplified DNA extracted from the blood of a volunteer infected with malaria.
  • the volunteer was infected on day 0 and samples taken on day 1 and then twice daily (day 2 to day 9, morning (am) and afternoon/evening (pm) sample). Duplicate assays at each time point are shown on this figure.
  • a trace of parasites were detected on day 1, day 2 am and pm samples, with a big increase in the amount of parasite DNA (upper band) in the pm sample from day 4.
  • Parasites were detected by microscopy on day 9.
  • Figure 5 is a graphical representation showing parasite growth curves for blood induced infections in volunteers, measured every 24 hours. Numbers in the legend represent the volunteers.
  • Anopheles stephensi mosquitoes were infected with P. falciparum clone 3D7A (a chloroquine sensitive line) by membrane feeding on a blood meal containing gametocytes.
  • the parasites were cultured in vitro under standard gametocyte culture conditions (13, 14), harvested after 14 and/or 7 days and mixed with fresh red blood cells and heat inactivated serum (supplied by the Edinburgh and South East Scotland Blood Transfusion Service, UK) to form the infectious blood meal.
  • Five day old mosquitoes, previously starved for 48 hours, were fed on this material through a membrane feeder (13, 14) and an uninfected blood meal was given 4 days later to increase the number of sporozoites in the salivary glands (15).
  • mosquitoes were fed on the volunteers until all, or nearly all, mosquitoes had taken a full blood meal. The fed mosquitoes were then killed with chloroform and dissected to assess the extent and level of sporozoite infection in the salivary glands.
  • volumenteer 1 On day zero, one volunteer (volunteer 1) was exposed to the bite of 22 mosquitoes of which 7 were sporozoite positive. On day 2, the volunteer was exposed to the bite of 3 more mosquitoes, 2 of which were sporozoite positive. Another volunteer (volunteer 2) was exposed to the bite of 25 mosquitoes on day zero of which 3 were sporozoite positive and a further 8 on day 2, 6 of which were sporozoite positive. Parasitaemia in both volunteers was followed daily by microscopy and PCR from day 4 to day 21 after infection.
  • a unit of blood (500 ml) was taken from both volunteers 6 hours after they became ill and developed high fever (13 and 14 days after the initial mosquito bites respectively) when both were microscopically positive.
  • the parasitaemia in the blood before processing was determined by counting the ratio of parasites to leucocytes on thick films, and using the measured leucocyte and red cell concentrations to calculate the proportion of red cells infected. The figure was used as the basis of calculating parasite inocula sizes.
  • Primers corresponding to the conserved regions in STEVOR were chosen to achieve high sensitivity.
  • the primers are shown in Table 1.
  • a particularly useful methodology employed a first round PCR using primers P5, P18, P19 and P20.
  • the DNA contained control plasmid DNA prior to total DNA extraction.
  • the first round 100 ⁇ l reaction included 20 ⁇ l of the 1 ml DNA solution isolated from blood; 150 ng of P5, P18, P20 and 200 ng of P19; 0.02 mM dNTPs (Promega, Madison, WI), 7.5 mM Mg ++ , 2.5U Taq polymerase (Perkin Elmer, Norwalk, CT) was performed for 22 cycles (93 °C, 30 sec, 50°C, 50 sec and 72°C, 30 sec) using a DNA Engine (MJ Research, Watertown, MS). Primers P17 were used in a 50 ⁇ l second round PCR.
  • PCR product Five to 10 ⁇ l of PCR product was loaded on a 3% w/v agarose (FMC BioProducts, Rockland, ME) gel and electrophoresed for 25 min at 120 volts. Following electrophoresis, a digital fluorescence image of the gel was captured with a NovaLine gel documentation system (Novex, Australia) and the relative fluorescence (F R ) in the parasite band compared with the total fluorescence (parasite+internal control) was calculated.
  • FMC BioProducts Rockland, ME
  • the sensitivity of amplification of STEVOR was compared to that of the gp 19 fragment of the single copy gene MSA-1.
  • the primers used are shown in Table 1.
  • the cycling conditions were: 95 °C 30 sec, 55 °C 50 sec and 72°C 30 sec for 40 cycles.
  • Five ⁇ l of the PCR product was loaded on a 3% w/v agarose gel containing 25 ng/ml ethidium bromide, electrophoresed and visualised on a UV transilluminator.
  • a fluorescence image of the gel was captured with a NovaLine gel documentation system (Novax). This uses a CCD camera with a HOYA HMC 46 mm O(G) filter to capture a digital image. The image was stored as a TLF file and the relative fluorescence of each band determined using ImageQuant software (Molecular Dynamics).
  • the primer pair P17 and P24 amplified a single band of 188bp from 0.002 pg of parasite DNA following one round of 40 cycles of amplification, which is equivalent to one tenth of a parasite ( Figure 2). This band was not observed when amplifying from uninfected human DNA or P. vivax infected samples. PCR of the MSA-1 gene detected 2pg of parasite DNA. This was 1000 fold less sensitive than the PCR of STEVOR ( Figure 2).
  • Figure 1 demonstrates the difference in sensitivity obtained using a single round of PCR with primers hybridising to the STEVOR sequences and to a single copy gene, PfMSP 1. Furthermore, the assay of the present invention amplified DNA from all 13 P. falciparum lines tested ( Figure 1) which indicates that the assay will be useful world wide.
  • Red blood cells infected with ring stage parasites of the 3D7 line of P. falciparum were added to 3 ml aliquots of freshly drawn human blood.
  • DNA was isolated from the 3 ml samples using a modified salting method originally described by La iri and Numberger (12). Samples were diluted to 10ml using lOmM Tris pH7.6, lOmM KC1, lOmM MgCl 2 and 2mM EDTA, and red blood cells lysed by adding lOO ⁇ l of 10% v/v saponin. Parasites were pelleted and washed with the same buffer twice (14,500xg for 15 min).
  • a volume of 1.6 ml of the above buffer containing 0.4M NaCl was added to the parasite pellet plus lOO ⁇ l of 20% w/v sodium dodecyl sulphate and incubated at 55 °C for 5-1 Omin. An amount of 0.0045pg of the plasmid DNA was added to the solution. After adding 0.6ml of 6M NaCl the samples were spun at 2400xg for 5 min. Supematants were transferred to fresh tubes and DNA precipitated by adding 2 vol of ethanol. After washing with 75% v/v ethanol DNA was air dried and redissolved in 1ml of water.
  • the assay can use internal control DNA for measuring the relative amount of parasite DNA in the blood sample.
  • one internal control molecule was engineered by cloning the 7H8/6 sequence with a deletion from the Bglll to AccI sites (from basses -13 to 99 and 154 to 300 numbered according to GeneBank sequence PFA7H86) into pUC18.
  • DNA from the M13 phage containing the 7H8/6 insert was amplified in a PCR with the P5 and P17 primers and the
  • Volunteer 1 received 3000 of the volunteer's stored infected red cells on day zero. Volunteers 2-5 received 3000, 3000, 6000 and 300 stored infected red cells, originally collected from volunteer 2. In this stored blood, there were 400 infected cells per ⁇ l of packed cells. All volunteers were followed at least daily by PCR and after day 6 of infection, by microscopy as well. Ten ml of venous blood was taken at 24 hour intervals for volunteer 2 and 12 hour intervals for the rest of the volunteers. Volunteers 1 to 3 were treated with chloroquine after they became positive by microscopy (day 8 to day 9) whereas volunteers 4 and 5 were treated when parasitaemia, measured by PCR, first reached 500 to 1000 parasites per ml.
  • the viability of thawed parasites was investigated by a limiting dilution assay followed by PCR.
  • the thawed inoculum was plated in 96-well plates at a theoretical 30, 10, 3, 1, 0.3 and 0.1 parasites/well based on post-thawed haematocrit counts and the microscopically determined parasitaemia prior to freezing.
  • 100 ⁇ l of culture medium containing 5 ⁇ l of uninfected red cells was added and the plates were incubated for 9 days.
  • Half of each culture was transferred to a new plate and lysed by adding 100 ⁇ l of 0.1 saponin in TKMl solution.
  • the growth rates and the effective starting parasitaemia were determined using a maximum likelihood fit to a model assuming that the inoculum had two broods of parasites separated by 24 hours and that parasites older than 24 hours would be sequestered.
  • this program allows estimates of growth rates where combination of synchronous parasites and sequestration results in decreased circulating parasitaemia on certain days.
  • all volunteers had a constant growth rate over the period of the infections of 15.5 ⁇ 2.8, 15.6 ⁇ 2.8, 13.0 ⁇ 3.0 and 17.2 ⁇ 3.2 fold per cycle (48 hours) for volunteers 2, 3, 4 and 5 respectively, with 39% of parasites dividing in the first 24 hours and the remainder over the second 24 hours.
  • the estimated starting parasitaemia derived from the curve fitting was 37% lower than the estimate based on both microscopic counts and on the number of viable parasites determined by limiting dilution in the inoculum.
  • all three measures have uncertainties.
  • errors in calculating the growth rates and starting parasitaemia are negatively correlated.
  • Growth rates calculated assuming a starting parasitaemia based on the microscopic estimate were 12.1 ⁇ 0.1, 12.2 ⁇ 0.1, 9.3 ⁇ 0.6 and 13.9 ⁇ 0.4 fold per cycle, respectively.
  • Volunteers 1, 2 and 3 were treated with chloroquine in the evening of day 8 (volunteers 1 and 2) and the morning of day 9 (volunteer 3) when symptoms occurred. Parasites were confirmed by thick film examinations in the three volunteers. Parasitaemia decreased rapidly after treatment. Volunteers 4 and 5 were treated on day 6 and 8, respectively, when parasitaemia first reach 500 to 1000 per ml. No symptoms were noted by these volunteers prior to and during the treatment.

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Abstract

L'invention concerne, d'une manière générale, le domaine des dosages à usage diagnostique et, plus particulièrement, une méthode de détection à usage diagnostique de Plasmodium falciparum, qui est le parasite responsable de la malaria. L'invention concerne, également, un ensemble de réactifs pour détecter P. falciparum. Grâce à l'invention, on peut, pour la première fois, doser par une méthode très sensible, le parasite de la malaria, ce qui est particulièrement utile pour déceler cette maladie particulièrement débilitante qu'est la malaria et suivre son traitement. Le dosage de la présente invention est particulièrement utile pour déterminer l'efficacité de vaccins contre la malaria.
PCT/AU1997/000076 1996-02-13 1997-02-12 Detection a usage diagnostique de plasmodium falciparum Ceased WO1997030176A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU15856/97A AU1585697A (en) 1996-02-13 1997-02-12 Diagnostic assay for plasmodium falciparum

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPN8037 1996-02-13
AUPN8037A AUPN803796A0 (en) 1996-02-13 1996-02-13 Diagnostic assay for plasmodium falciparum

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WO1997030176A1 true WO1997030176A1 (fr) 1997-08-21

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020028385A (ko) * 2000-10-09 2002-04-17 박제철 마커를 이용한 말라리아 원충에 감염된 모기 검출방법
WO2006004332A1 (fr) * 2004-06-30 2006-01-12 Lg Life Sciences Ltd. Dosage immunologique pour plasmodium falciparum et dispositif de dosage utilise a cette fin
CN110358853A (zh) * 2019-07-20 2019-10-22 昆明医科大学 一种检测恶性疟原虫dhps基因点突变的方法及其试剂盒

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AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, Volume 51, (1994), ARAI et al., "Detection of Plasmodium Falciparum in Human Blood by a Nested Polymeric Chain Reaction", pages 617-626. *
CELL, Volume 53, (1993), CORCORAN et al., "Homologous Recombination Within Subtelomeric Repeat Sequences Generates Chromosome Size Polymorphums in P. Falciparum", pages 807-813. *
CELL, Volume 55, (1988), POLONGE et al., "Large Deletions Result from Breakage and Healing of P. Falciparum Chromosomes", pages 869-874. *
GENOMICS, Volume 26, (1995), RUBIO et al., "A YAC Contig Map of Plasmodium Falciparum Chromosome 4: Characterization of a DNA Amplification Between Two Recently Seperated Isolates", pages 192-198. *
JOURNAL OF MOLECULAR BIOLOGY, Volume 185, (1985), ASLUND et al., "Highly Reiterated Non-Coding Sequence in the Genome of Plasmodium Falciparum is Composed of 21 Base-Pair Tandem Repeats", pages 509-516. *
MOLECULAR AND BIOCHEMICAL PARASITOLOGY, Volume 47, (1991), LIMPAIBOON et al., "7H8/6, a Multicopy DNA Probe for Distinguishing Isolates of Plasmodium Falciparum", pages 197-206. *
MOLECULAR AND BIOCHEMICAL PARASITOLOGY, Volume 58, (1993), SCOTTI et al., "A 40-Kilobase Subtelomeric Region is Common to Most Plasmodium Falciparum 3D7 Chromosomes", pages 1-6. *
MOLECULAR AND BIOCHEMICAL PARASITOLOGY, Volume 61, (1993), DOLAN et al., "Restriction Polymorphisms and Fingerprint Patterns from an Interspersed Repetitive Element of Plasmodium Falciparum DNA", pages 137-142. *
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PROC. NATL. ACAD. SCI. U.S.A., Volume 86, (1989), BIGGS et al., "Subtelomeric Chromosome Deletions in Field Isolates of Plasmodium Falciparum and Their Relationship to Loss of Cytoadherence in Vitro", pages 2428-2432. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 91, (1994), DE BRUIN et al., "The Polymorphic Subtelomeric Regions of Plasmodium Falciparum Chromosomes Contain Arrays of Repetitive Sequence Elements", pages 619-623. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020028385A (ko) * 2000-10-09 2002-04-17 박제철 마커를 이용한 말라리아 원충에 감염된 모기 검출방법
WO2006004332A1 (fr) * 2004-06-30 2006-01-12 Lg Life Sciences Ltd. Dosage immunologique pour plasmodium falciparum et dispositif de dosage utilise a cette fin
KR101035111B1 (ko) * 2004-06-30 2011-05-19 주식회사 엘지생명과학 말라리아 플라스모듐 팔시파룸의 면역학적 측정방법 및이에 사용되는 측정 수단
CN110358853A (zh) * 2019-07-20 2019-10-22 昆明医科大学 一种检测恶性疟原虫dhps基因点突变的方法及其试剂盒
CN110358853B (zh) * 2019-07-20 2023-04-07 昆明医科大学 一种检测恶性疟原虫dhps基因点突变的方法及其试剂盒

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