Nucleic acid hybridization assays employing dA-tailed capture probes

Hunsaker, William R.; Badri, Hummy; Lombardo, Massimo; Collins, Mark L.

doi:10.1016/0003-2697(89)90256-x

Cited by 31 publications

(12 citation statements)

References 28 publications

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“…Moreover, the RNA obtained by these methods is also contaminated with >70% of 16S and 23S rRNAs [6]. Removal of contaminating rRNA often requires an extra purification step using nucleic acid capture techniques [7–9]. The Qiagen and hot phenol extraction methods yielded RNA that was contaminated with 16S and 23S rRNA (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The rRNA and tRNA are of no use in various molecular biology applications such as cloning, reverse transcription (RT)/RT‐polymerase chain reaction (PCR), and gene‐expression assays. To free bacterial RNA preparations from contaminating rRNA, modified versions of nucleic acid capture techniques [7–9] are used. These involve binding of rRNA to a capture oligonucleotide that contains sequences complementary to 16S and 23S rRNA at one end and sequences at the other end complementary to the sequences attached to magnetic beads.…”

Section: Introductionmentioning

confidence: 99%

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A simple and efficient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria

Sung

Khan

Nawaz

et al. 2003

FEMS Microbiology Letters

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A fast, reliable, and inexpensive Triton X-100 boiling procedure for RNA isolation from both the Gram-positive and Gram-negative bacteria was developed. The yield of RNA was 0.2-2 mg per 10 ml bacterial culture. The method was tested on Gram-positive and Gram-negative bacteria of eight genera and nine species and yielded reproducible results. In parallel experiments, the Qiagen and hot phenol extraction methods both yielded RNA that contained contaminating 16S and 23S rRNA. The Triton X-100 boiling method reported here yielded RNA that was free from 16S and 23S rRNA, contained full-length transcripts and did not require additional purification. The presence of specific mRNA in one of the RNA samples obtained by this procedure was demonstrated by partial amplification of a 732 bp vancomycin resistance gene, vanA, by reverse transcription-polymerase chain reaction (RT-PCR). The presence of a full-length transcript (1031 bases) of the vanA gene was verified by Northern hybridization and probing with a digoxigenin (DIG)-labeled vanA PCR partial product. The method provides a rapid, reliable, and simple tool for the isolation of good quality RNA suitable for various molecular biology experiments.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A simple and efficient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria

Sung

Khan

Nawaz

et al. 2003

FEMS Microbiology Letters

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“…DNA fragments or PCR products larger than 300 bp can be coated onto a solid phase either covalently (15) or by passive adsorption (13,17). Oligo(dT)14 covalently linked to the solid phase was used to capture a poly(A) tail (12,27).…”

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confidence: 99%

Enzyme-linked oligosorbent assay for detection of polymerase chain reaction-amplified human immunodeficiency virus type 1

et al. 1993

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An enzyme-linked oligosorbent assay (ELOSA) was developed for the detection on microtiter plates of polymerase chain reaction (PCR)-amplified human immunodeficiency virus type 1 (HIV-1) DNA. The denatured PCR product was hybridized with a passively adsorbed oligonucleotide capture probe and a horseradish peroxidase-labeled oligonucleotide detection probe. The sensitivity and specificity of the PCR-ELOSA technique depended to some extent on the nucleotide sequences of the oligonucleotide primer and probe quartet used in the amplification and detection. We evaluated five oligonucleotide quartets located in the gag, pol, vpr, env, and nef regions of HIV-1. DNAs from 39 HIV-1-seropositive individuals and 27 healthy HIV-1-seronegative controls were amplified by the PCR procedure, and the products were detected by ELOSA. Ten copies of HIV-1 DNA against a background of 1 ,ug of human DNA were specifically detected by PCR-ELOSA. Specificities and sensitivities were, respectively, 100 and 95% for the gag system, 100 and 97% for the pol system, 100 and 85% for the vpr system, 96 and 95% for the env system, and 100 and 95% for the nef system. The simplicity of ELOSA makes it suitable for automation and applicable to genetic testing and detection of viral and bacterial DNAs or RNAs in most routine laboratories.

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“…Paramagnetic particles were obtained from Advanced Magnetics, Inc. (Cambridge, Mass.). The particles were derivatized with either oligo(dT) 14 or oligo(dC) 25 as previously described (9,21,22,25).…”

Section: Methodsmentioning

confidence: 99%

Comparison of amplified Q beta replicase and PCR assays for detection of Mycobacterium tuberculosis

Buxton

Hendricks

et al. 1995

J Clin Microbiol

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Because of the long time required to isolate Mycobacterium tuberculosis in culture, there is an acute need for simple rapid methods for direct detection of M. tuberculosis from human sputum specimens. We have developed and characterized quantitative manual Q␤ replicase and PCR assays for M. tuberculosis. The Q␤ replicase assay was based on reversible target capture of M. tuberculosis 23S rRNA followed by amplification of a replicatable detector probe with Q␤ replicase. For PCR assays, primers generating a 370-bp amplification product from the IS6110 insertion element were used in combination with a control plasmid containing an internal deletion in the IS6110 amplicon. Serial dilutions of M. tuberculosis were spiked into sputum and subjected to digestion and decontamination with N-acetyl-L-cysteine and NaOH. Assay conditions were optimized for hybridization and sample processing chemistries in order to maximize sample utilization. Following assay optimization, the sensitivities of the Q␤ replicase and PCR assays of spiked sputum samples were 0.5 and 5.0 CFU per assay reaction, respectively. The effects of sputum matrix on each assay were examined by testing 20 patient sputum samples which had been cultured for M. tuberculosis. The culture-positive samples included smear-positive and smear-negative samples. The results of the Q␤ replicase assay were not inhibited by sputum and were in 100% agreement with those of culture, including detection of 10 culture-positive specimens. However, using an internal control plasmid coamplified with each PCR as an indicator, we detected PCR inhibition in 9 of 20 samples tested. Decreasing the amount of sample assayed in the PCR 24-fold alleviated the inhibitory effects in all but two specimens, one of which was culture positive. The decreased sample utilization also resulted in a false-negative result with a third specimen which was culture positive for M. tuberculosis. Quantitative smear results and Q␤ replicase assay estimates of the number of organisms present in these specimens were in close agreement. The Q␤ replicase assay performed well in comparison with both culture and PCR and should offer a rapid means for detecting and controlling infection due to M. tuberculosis.

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Nucleic acid hybridization assays employing dA-tailed capture probes

Cited by 31 publications

References 28 publications

A simple and efficient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria

A simple and efficient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria

Enzyme-linked oligosorbent assay for detection of polymerase chain reaction-amplified human immunodeficiency virus type 1

Comparison of amplified Q beta replicase and PCR assays for detection of Mycobacterium tuberculosis

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