A polymerase chain reaction assay for the detection of Brugia malayi in blood.
There is need for sensitive, rapid, species-specific diagnosis of Brugia filarial parasites because traditional methods are tedious and time-consuming, with little guarantee of species specificity. A polymerase chain reaction (PCR)-based assay was developed using the Hha I family of highly repeated DNA sequences from Brugia. The assay was tested on 124 human blood samples collected in a field study in Indonesia. These included 66 microfilaria-positive samples from patients in an area endemic for Brugia, 30 from healthy individuals from the same endemic area, and 28 from healthy individuals from a nonendemic area. Twenty-eight blood samples collected in a village in French Polynesia endemic for Wuchereria bancrofti, but not B. malayi, were also tested. The blood samples were screened using the traditional blood smear and membrane filtration methods, which served as the gold standards to which the PCR assay was compared. The samples were digested with proteinase K, extracted with phenol and chloroform, and dialyzed. A fraction of the dialyzed product was used in PCRs using Hha I-specific primers. The PCR assay correctly identified all of the microfilaria-positive samples as PCR positive and all of the nonendemic samples as PCR negative. Additionally, 26 of 30 samples from healthy individuals in the endemic area were also identified as PCR negative, while four were PCR positive. It is likely that these four individuals had very low-level or cryptic infections, and that the PCR assay detected circulating DNA released from dead filariae. The results indicate that the Hha I PCR detection system is rapid, species-specific, and sensitive.
Lizotte
M R
MR
Department of Biological Sciences, Smith College, Northampton, Massachusetts.
Supali
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Partono
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Williams
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SA
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D017178:Brugia malayi / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry; D018509:DNA, Helminth / Q000097:blood* / Q000737:chemistry; D015257:DNA-Cytosine Methylases; D005368:Filariasis / Q000175:diagnosis*; D006801:Humans; D008842:Microfilariae / Q000302:isolation & purification; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013997:Time Factors
D017931:DNA Primers; D018509:DNA, Helminth; D015345:Oligonucleotide Probes; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases