Detection of viable Giardia cysts by amplification of heat shock-induced mRNA

Abbaszadegan, Morteza; Huber, Mary S.; Gerba, Charles P.; Pepper, Ian L.

doi:10.1128/aem.63.1.324-328.1997

Cited by 41 publications

(8 citation statements)

References 29 publications

(30 reference statements)

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“…In particular, PCR is an attractive diagnostic procedure as it is rapid, sensitive, and pathogen specific. While many PCR methods have been described for both Giardia and Cryptosporidium detection (1,11,18,20,23,24,26,29,39,47), this technology is only slowly emerging as a practical method for pathogen assessment of water quality. This is largely due to the low numbers of cysts and oocysts in water and the requirement for significant sample concentration factors to reduce large sample volumes to the microliter quantities that are compatible with PCR.…”

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confidence: 99%

Sensitive and Rapid Detection of Viable Giardia Cysts and Cryptosporidium parvum Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR

Kaucner¹,

Stinear²

1998

Appl Environ Microbiol

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We recently described a reverse transcription-PCR (RT-PCR) for detecting low numbers of viable Cryptosporidium parvumoocysts spiked into clarified environmental water concentrates. We have now modified the assay for direct analysis of primary sample concentrates with simultaneous detection of viable C. parvum oocysts, Giardia cysts, and a novel type of internal positive control (IPC). The IPC was designed to assess both efficiency of mRNA isolation and potential RT-PCR inhibition. Sensitivity testing showed that low numbers of organisms, in the range of a single viable cyst and oocyst, could be detected when spiked into 100-μl packed pellet volumes of concentrates from creek and river water samples. The RT-PCR was compared with an immunofluorescence (IF) assay by analyzing 29 nonspiked environmental water samples. Sample volumes of 20 to 1,500 liters were concentrated with a wound fiberglass cartridge filter. Frequency of detection for viable Giardiacysts increased from 24% by IF microscopy to 69% by RT-PCR. ViableC. parvum oocysts were detected only once by RT-PCR (3%) in contrast to detection of viable Cryptosporidium spp. in four samples by IF microscopy (14%), suggesting thatCryptosporidium species other than C. parvumwere present in the water. This combination of the large-volume sampling method with RT-PCR represents a significant advance in terms of protozoan pathogen monitoring and in the wider application of PCR technology to this field of microbiology.

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confidence: 99%

Sensitive and Rapid Detection of Viable Giardia Cysts and Cryptosporidium parvum Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR

Kaucner¹,

Stinear²

1998

Appl Environ Microbiol

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“…Abbaszadegan and colleagues (1991) detected Giardia in water samples by a cDNA probe [28]. In subsequent studies, the sensitivity level increased to detection of a DNA equivalent to a single Giardia cyst by a PCR assay [29]. The advantages of PCR technique included the distinction between live and dead cysts as well as identification of, and differentiation between Giardia species [30,31].…”

Section: Discussionmentioning

confidence: 99%

Detection of Giardia lamblia Cysts in Surface Waters of Rasht City, Iran

saeedi

Jafari

Salehzadeh

2018

JoMMID

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“…Thus, determining how well this RT-PCR assay assesses cyst viability is key to its use with environmental samples. There are precedents for use of the RT-PCR in assessing the viability of both Giardia cysts (1,19) and Cryptosporidium oocysts collected from environmental samples (19). These assays all exploited the heat shock responses of the two organisms, distinguishing viable from nonviable cysts by detecting the heat shock transcript by RT-PCR after heat treatment.…”

Section: Discussionmentioning

confidence: 99%

Development and Evaluation of a Molecular Viability Assay for Pneumocystis carinii

et al. 2000

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Despite recent declines in incidence, Pneumocystis carinii pneumonia (PCP) remains the most commonly occurring opportunistic illness among persons with AIDS in the United States. While P. carinii DNA has been detected in patient respiratory specimens and in air samples collected from various indoor environments housing PCP patients, the viability of these organisms is unknown. For this reason, we have developed and evaluated a molecular viability assay for P. carinii. This method is based upon the detection of P. carinii mRNA by a reverse transcription-PCR that employs specific primers from a member of the heat shock protein 70 family. Under optimal assay conditions, these primers were capable of detecting as few as 100 viable trophozoites as determined by ethidium bromide staining, while no signal was obtained from 106 trophozoites killed by heat, desiccation, or UV radiation. This assay was also capable of distinguishing P. carinii from other common fungi present in the air. Therefore, this molecular viability assay may be useful in conjunction with standard bioaerosol collection devices and procedures for the detection of viable P. carinii collected from various indoor environments. It may also be useful in confirming the presence of viable trophozoites in respiratory specimens collected by noninvasive techniques from putatively infected individuals.

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Detection of viable Giardia cysts by amplification of heat shock-induced mRNA

Cited by 41 publications

References 29 publications

Sensitive and Rapid Detection of Viable Giardia Cysts and Cryptosporidium parvum Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR

Sensitive and Rapid Detection of Viable Giardia Cysts and Cryptosporidium parvum Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR

Detection of Giardia lamblia Cysts in Surface Waters of Rasht City, Iran

Development and Evaluation of a Molecular Viability Assay for Pneumocystis carinii

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