Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (approximately375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects approximately550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, approximately2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp(R) DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer's protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit's protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 microl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, approximately 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.

Isolation and Cloning of an ABC Transporter-Like Gene of Haemophilus parasuis and Its Use in a New Diagnostic PCR

The aim of this study was to identify a new gene of Haemophilus parasuis that could be used to develop a polymerase chain reaction (PCR) test for this porcine pathogen. H. parasuis genomic DNA was cloned into a set of expression vectors, and transformants expressing His-tagged polypeptides were identified by colony blotting. An ABC transporter-like gene was isolated. The cloned DNA fragment is 1,105 base pair and shows 78% similarity at the nucleotide level with an ABC transporter gene of H. ducreyi. Based on this sequence, two PCR primers were designed to amplify the entire 1,105-bp fragment in the proposed diagnostic PCR test. PCR amplification was able to detect a minimum of 1 x 10(4) CFU/ml of H. parasuis organisms. Fifteen different H. parasuis serovars were positive using the PCR test. No amplification was observed when the test was done using DNA from 16 other bacterial species commonly isolated from swine.

Authentication of Dendrobium nobile by allele-specific diagnostic PCR / 中草药

Objective To develop a convenient and effective molecular authentication method for identifying Dendrobium nobile from other species of Dendrobium Sw.Methods Based on rDNA ITS sequences of D.nobile and other species of Dendrobium Sw.of Huangcaos and Fengdous,unusual sequence sites were confirmed and an allele-specific diagnostic primer pair was designed for D.nobile. Diagnostic PCR was performed using the primers with the total DNA of D.nobile and other species of Dendrobium Sw.as templates to establish the positive PCR condition for D.nobile.Results An about 300 bp DNA positive fragment was amplified from D.nobile with anneal temperature at 66 ℃ and anneal time at 40 s,whereas no any DNA fragment was amplified from the rest 39 species of Dendrobium Sw.Conclusion D.nobile could be effectively distinguished from other species of Dendrobium Sw.by diagnostic PCR with allele-specific diagnostic PCR,which is not only effective and specific,but also simple and timesaving,and has a comprehensive application prospects.

Allele-specific diagnostic PCR authentication of Dendrobium huoshanense and its allied species of Dendrobium Sw / 中草药

Objective To design a pair of allele-specific diagnostic primer for authenticating Dendrobium huoshanense from other species of Dendrobium Sw.only by PCR.Methods Based on rDNA ITS sequences database of D.huoshanense and other species of Dendrobium Sw.,an allele-specific diagnostic primer pairs was designed.Diagnostic PCRs were performed using the primer with the total DNAs of 19(original) plants from Dendrobium Sw.as templates.The positive was the genuine.Results When the(annealing) temperature was raised to 60 ℃, the template DNA of D.huoshanense could be amplified whereas the diagnostic PCR of the rest species were all negative.Conclusion The diagnostic PCRs can authenticate D.huoshanense from other species of Dendrobium Sw.efficiently.Compared to the authentication method by sequencing DNA fragments and morphological traits etc.,the allele-specific diagnostic PCR is not only simple with time-saving but also practical and effective.