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Probetec strand displacement amplification ppt

probetec strand displacement amplification ppt

The BD ProbeTec CT Qx Amplified DNA Assay and the BD ProbeTec GC Qx real-time strand displacement amplification (SDA)/detection of CT. The performance of nucleic acid amplification tests (NAATs) with and both Becton Dickinson tests use strand displacement amplification. PCR Platform for Multiple Sexually Transmitted Diseases Strand Detection such as ELISA, (7) strand displacement amplification. SCIENCE HAS MADE THE WORLD A BETTER PLACE TO LIVE IN

Certain false positives and false negatives can occur as a consequence of specimen collection, test operation, and laboratory environment. The increment for detection of gonococcal infections is somewhat less. Detection of Genitourinary C. The prevalence of gonorrhea varies widely among communities and populations; health-care providers should consider local gonorrhea epidemiology when making screening decisions. Although widespread screening is not recommended, targeted screening of young women i.

For sexually active women, including those who are pregnant, the U. For female screening, specimens obtained with a vaginal swab are the preferred specimen type. Vaginal swab specimens are as sensitive as cervical swab specimens, and there is no difference in specificity 82— Self-collected vaginal swabs are equivalent in sensitivity and specificity to those collected by a clinician 83, Cervical samples are acceptable when pelvic examinations are done, but vaginal swab specimens are an appropriate sample type, even when a full pelvic exam is being performed.

However, following Pap screening, there should be a clinical indication for reflex additional testing of liquid cytology specimens for chlamydia and gonorrhea since these specimen types are more widely used in older populations at low risk for infection. Although chlamydia prevalence data have provided a basis for setting age guidelines for routine annual screening and behavioral guidelines for targeted screening in women 11 , no such consensus has been reached regarding control program definitions in men who have sex with women Although there are no recommendations to screen heterosexual men, it USPSTF suggests testing to test sexually active heterosexual men in clinical settings with a high prevalence of C.

The prevalence of N. There is insufficient evidence for or against routine screening for gonorrhea in sexually active heterosexual men at increased risk for infection However, it is not recommended to screen for gonorrhea infections in men at low risk for infection Overwhelming evidence described the performance of male first catch urine samples as equivalent to, and in some situations superior to, urethral swabs 23, Use of urine samples is highly acceptable and might improve the likelihood of uptake of routine screening in men Box 3.

Detection of Extragenital C. Because extragenital infections are common in MSM, and most infections are asymptomatic 91 , routine annual screening of extragenital sites in MSM is recommended. No recommendations exist regarding routine extragenital screening in women because studies have focused on genitourinary screening, but rectal and oropharyngeal infections are not uncommon.

The scope of the problem of extragenital infection in MSM is not known at the national level. In , CDC coordinated an evaluation of MSM attending several community-based organizations and public or STD clinics and found that of approximately 30, tests performed, 5.

Pharyngeal N. For oropharyngeal N. For oropharyngeal infections with C. The number of infections detected was more than doubled when a more sensitive NAAT was used as compared with the use of standard culture. Other researchers also have demonstrated the superiority of NAATs as compared with culture for diagnosing C. Results from commercially available NAATs can be used for patient management if the laboratory has established specifications for the performance characteristics according to CLIA regulations If a moderate complexity test such as the GeneXpert is modified in any manner, the test defaults to high complexity and the laboratory must meet all high complexity CLIA requirements, including those for personnel.

Certain NAATs that have been demonstrated to detect commensal Neisseria species in urogenital specimens might have comparable low specificity when testing oropharyngeal specimens for N. Thus, a N. Detection of Genitourinary and Extragenital C.

General recommendations pertaining only to C. Examination of victims is required for two purposes: 1 to determine if an infection is present so that it can be successfully treated and 2 to acquire evidence for potential use in a legal investigation. Testing to satisfy the first purpose requires a method that is highly sensitive, whereas satisfying the second purpose requires a method that is highly specific. Although NAATs meet these criteria, acceptance of any test results is determined by local legal authorities.

Local legal requirements and guidance also should be sought for maintaining and documenting a chain of custody for specimens and results that might be used in a legal investigation and for which test results are accepted as evidence. NAATs for C. Consultation with an expert is necessary before use of NAATs for this indication in children to minimize the possibility of positive reactions with nongonococcal Neisseria species and other commensals.

NAATs can be used as alternative to culture with vaginal specimens or urine specimens from girls. Culture remains the preferred method for urethral specimens from boys and extragenital specimens pharynx and rectum in boys and girls. Using highly specific tests is critical with prepubescent children for whom the diagnosis of a sexually transmitted infection might lead to initiation of an investigation for child abuse. Specimen collection for culture for N.

Cervical specimens are not recommended for prepubertal girls. For boys with a urethral discharge, a meatal specimen discharge is an adequate substitute for an intra-urethral swab specimen. Standard culture procedures must be followed. Gram stains are inadequate to evaluate prepubertal children for N. Specimens from the vagina, urethra, pharynx, or rectum should be streaked onto selective media for isolation of N. Isolates should be preserved to enable additional or repeated testing.

Cultures for C. The likelihood of recovering C. However a meatal specimen should be obtained if urethral discharge is present. Pharyngeal specimens for C. All specimens must be retained for additional testing, if necessary, regardless of a positive or negative test result. Only standard culture systems for the isolation of C.

The isolation of C. EIAs are not acceptable confirmatory methods. Isolates should be preserved. Nonculture tests for C. NAATs can be used for detection of C. No data exist on the use of nucleic acid amplification tests in boys and extragenital specimens rectum in boys and girls. Culture remains the preferred method for extragenital sites in these circumstances.

The chlamydial complement fixation test CFT , which measures antibody against group-specific lipopolysaccharide antigen, has been used as an aid in the diagnosis of LGV. The micro-immunofluorescence MIF test was initially developed for serotyping strains of C. Although the original MIF method was complicated, involving the titration of sera against numerous antigens, it was found to have many advantages when compared with the CFT The MIF test can be used detect type-specific antibody and different immunoglobulin classes.

The MIF test is more sensitive than the CFT with a larger proportion of patients developing an antibody response and at higher titer. Microtiter plate format enzyme immunoassays have been developed but comparative performance data are lacking. Serologic test interpretation for LGV is not standardized, tests have not been validated for clinical proctitis presentations, and C. More detailed information concerning the diagnosis and treatment of LGV has been published Genital and lymph node specimens i.

Additional molecular procedures e. For patients presenting with proctitis, C. While a positive result is not definitive diagnosis of LGV, the result might aid in a presumptive clinical diagnosis of LGV proctitis. This was particularly important when the test was used in a population with a low prevalence of infection. When these NAATs are used, consideration should be given to retest these specimens with an alternate target assay if the anatomic site from which the specimen was collected is typically colonized with these commensal organisms, e.

As with any diagnostic test, if there is a clinical or laboratory reason to question a test result, a repeat test should be considered. Test interpretation. The laboratory should interpret and report results according to the manufacturer's package insert instructions In the event of discordant results from multiple tests, the report should indicate the results of both the initial and any additional tests.

An interpretation of "inconclusive," "equivocal," or "indeterminate" would be most appropriate. A new specimen should be requested for testing in these situations. All test results should be interpreted by clinicians within the context of the patient-specific information to determine appropriate patient management.

Test of cure. Culture is the only method that can be used to properly assess the efficacy of antibiotic therapy because commercial NAATs are not FDA-cleared for use as a test of cure. Residual nucleic acid from bacteria rendered noninfective by antibiotics might still give a positive C. Detection of N.

Pooling of specimens. Samples of individual specimens are first combined into a pool, which is then tested by a NAAT. If the pool is negative, all specimens forming the pool are reported as negative. If the pool is positive, a second aliquot of each specimen that contributed to the pool is tested individually. The potential cost-savings with pooling increases with decreasing prevalence of infection, because more specimens can be included in a pool without increasing the probability of a pool testing positive.

The number of specimens pooled to achieve the greatest cost savings for a particular prevalence can be calculated Available evidence indicates that pooled aliquots from up to 10 urine specimens can be a cost-effective alternative to testing individual specimens without any loss of sensitivity or specificity However, the increased complexity of the pooling protocol might require more personnel time to deconstruct positive pools for individual specimen testing.

The use of pooled specimens for testing is not cleared by FDA and, therefore, the CLIA requirements applicable to modifying a test procedure must be met before implementation and reporting results intended to guide patient care. Laboratories must be aware that the process of pooling specimens requires extensive handling of samples which increases the potential for cross-contamination. Studies for pooling clinical specimens other than urine are required before extending this recommendation.

This assay should not be used for routine testing of genital tract specimens. Depending on the commercial product used, the antigen that is detected by the antibody in the C. Specimen material is obtained with a swab or endocervical brush, which is then rolled over the specimen well of a slide. After the slide has dried and the fixative applied, the slide can be stored or shipped at ambient temperature.

Staining consists of flooding the smear with fluorescein-labeled monoclonal antibody that binds to C. Stained elementary bodies are then identified by fluorescence microscopy. Only C. The procedure requires an experienced microscopist and is labor-intensive and time-consuming.

No DFA tests exist for the direct detection of N. Two nucleic acid hybridization assays are FDA-cleared to detect C. Nucleic acid genetic transformation tests. The test has received limited evaluation in published studies — , which included an evaluation of its use with mailed specimens Amplified and hybridization tests that detect N. The gonorrhea nucleic acid genetic transformation test might have some utility in settings that lack the stringency for gonorrhea culture.

However, it is not recommended as an alternative test to N. A genetic transformation test is not available for detection of C. Enzyme immunoassay EIA tests. A substantial number of EIA tests have been marketed for detecting C. The performance and cost characteristics of EIA tests for N.

Manufacturers have developed blocking assays that verify positive EIA test results to improve specificity. Serology tests. Serology has little, if any, value in testing for uncomplicated genital C. It should not be used for screening because previous chlamydial infection might or might not elicit a systemic antibody response. Infections caused by LGV serovars of C. The complement fixation test was classically used for this purpose but has been replaced by the more sensitive species-specific microimmunofluorescence test.

A serologic screening or diagnostic assay is not available for N. Conclusion Technological evolution in clinical laboratory diagnostics has advanced considerably by allowing for the direct molecular detection of a pathogen in a clinical specimen rather than relying on isolation and cultivation. This approach has decreased the time required to identify a pathogen because the laboratory is no longer limited by the growth kinetics of the organism.

Therefore, patients can be evaluated and if infected can be treated promptly, thereby diminishing progression to disease and disrupting transmission. As with all changes in laboratory technology, a synthesis of scientific evidence is required for an informed decision regarding the implementation of a new or improved test platform. These updated CDC recommendations now specify that vaginal swabs are the preferred specimen for screening women and include the use of rectal and oropharyngeal specimens among populations at risk for extragenital tract infections.

FDA clearance is important for widespread use of a test, and it is important that clearance be obtained for NAAT use with rectal and oropharyngeal specimens, and with vaginal swabs collected in other than clinic settings. Future revisions to these recommendations will be influenced by the development and marketing of new laboratory tests, or indications of existing tests, for chlamydia and gonorrhea.

Improvements in molecular tests that continue to decrease detection time and decrease the test complexity might facilitate the use of NAATs in non-traditional laboratory settings such as physician offices, health fairs, school clinics, or other outreach venues. Shifting chlamydia and gonorrhea diagnostics from laboratories might require new recommendations on test application or reporting positive cases of reportable diseases. References CDC. Sexually transmitted disease surveillance, Stamm WE.

Chlamydia trachomatis infections of the adult. Sexually transmitted diseases. The estimated direct medical cost of selected sexually transmitted infections in the United States, Sex Transm Dis ;— Trends in the diagnosis and treatment of ectopic pregnancy in the United States. Obstet Gynecol ;— Productivity losses attributable to untreated chlamydial infection and associated pelvic inflammatory disease in reproductive-aged women.

Sex Transm Dis ;33 Suppl :S— Effect of treatment regimens for Neisseria gonorrhoeae on simultaneous infection with Chlamydia trachomatis. N Engl J Med ;—9. Rees E. Treatment of pelvic inflammatory disease. Am J Obstet Gynecol ;—7. Pelvic inflammatory disease and fertility: a cohort study of 1, women with laparoscopically verified disease and control women with normal laparoscopy results. The association between Chlamydia trachomatis and ectopic pregnancy: a matched-pair, case-control study.

JAMA ;—7. Serologic evidence for the role of Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma hominis in the etiology of tubal factor infertility and ectopic pregnancy. Sex Transm Dis ;—4. Screening for chlamydial infection: recommendation statement. Ann Intern Med ;— Male chlamydia screening consultation, Atlanta, Georgia, March 28—29, Meeting Report, May 22, Clinical features of Chlamydia trachomatis rectal infection by serovar among homosexually active men. Sex Transm Dis;—8.

Two target-specific aptamers termed biotinylated capture aptamers C-aptamer and amplifying aptamers A-aptamer bind to target cells, for the enrichment of targeted cells and strand displacement amplification SDA , respectively. After the binding reaction, streptavidin SA -coated magnetic beads are added to form cell-magnetic bead-aptamer complexes that are collected for SDA amplification.

Finally, the single strand amplicons are loaded onto the lateral flow strip for visual readout or hybridized to fluorophore-probe after displacement of quencher-labeled probe for fluorescent detection. Advertisement 3. SDA product analysis Detection of amplified product is critical in isothermal amplification of nucleic acids, which includes quantification of the final amplification product and monitoring of the product during the process of amplification.

Fluorescent dye-based analysis Fluorescent gel staining dyes such as SYBR Green and ethidium bromide are commonly used for monitoring and quantitation of nucleic acids. Thus, other dyes such as Picogreen, Ribogreen, and Oligogreen have been developed.

These versatile fluorescent dyes are specific, sensitive, and rapid for a broad spectrum of applications such as nucleic acid typing, amplification, and purification, Band-shift assays as well as DNA-damage assays. Interestingly, they are not interfered by free nucleotides and proteins.

They are much more specific than UV-based absorbance A and much easier to use than laborious radioisotope labeling and silver staining. They have been widely applied to quantitatively detect the early onset of diseases with window periods such as virus, oncogenes, etc. Furthermore, DNA dyes such as EMA ethidium monoazide bromide azide and PMA propidium monoazide bromide azide have been extensively used to distinguish nucleic acids of live and dead cells [ 63 , 64 ].

These dyes can penetrate damaged cells and bind to DNA with little effect on live cells endowed from their intact cell membranes [ 65 , 66 ]. However, their off-target binding and background result in spurious amplicon staining [ 47 , 50 ] and are limited to single-strand conformation polymorphism not at the level of single nucleotide polymorphism [ 67 ]. Fluorescent probe-based analysis To increase specificity, fluorescent oligo probes such as TaqMan probes and molecular beacons have been used to monitor and quantify nucleic acid amplification products [ 68 , 69 , 70 ].

There are two main types of fluorescent oligo probes, TaqMan probes and molecular beacons. In TaqMan probes, the fluorescent light of the fluorophore e. As a result, the fluorescent reporter and the quencher are separated, and the fluorescent reporter is then detected [ 71 ]. This technology has been widely used in real time PCR for medical diagnosis.

TaqMan probes depend on probe hybridization, polymerase extension, and cleavage of the probes. Molecular beacons, on the other hand, do not require polymerase extension and cleavage activity. Before hybridization with the target sequence, the fluorophore on one end of the molecular beacon is quenched by the quencher on the other end of the beacon as the two ends are close together.

When the probe hybridizes with the target nucleic acid sequence, the molecular beacon sequence becomes linear. As a result, the fluorophore and the quencher are separated, and the fluorescence is then detected. It can also been multiplexed using multicolored molecular beacons for different targets [ 69 ].

Organic dyes such as rhodamine are conventionally used in fluorescent oligo probes. However, organic dyes have low quantum yield and are easily photo bleached. Other fluorophores such as quantum dots QDs , silver nanoclusters, upconversion nanoparticles, and corresponding quenchers such as gold nanoparticles and carbon nanomaterials have been used to replace organic fluorophores and quenchers [ 73 ] for nuclei acid as well as protein detection [ 74 , 75 ].

These nanomaterials have high quantum yield and photostability. In addition, they can be simultaneously excited using one wavelength during multiplex detection of various targets. They are promising substitutes of organic dyes in the detection of nucleic acid detection using fluorescent oligo probes.

Tavares et al. Silver nanoclusters possess much higher photostability and fluorescence than organic fluorophores and QDs, which have been used to detect influenza specific nucleic acids. Upon hybridization, these DNA-silver nanocluster probes fluoresce up to fold when placed near G-rich nucleic acid targets and exhibited high signal to background ratio [ 77 ].

This finding was promising; however, it was elusive how fluorescence increased upon G-rich target detection. It is speculated that upon target binding guanines, G-quadruplex structures may be formed to yield reddish nanoclusters, or serve as electron donors since guanines have lowest oxidation potential compared with other nucleotides [ 78 ], or otherwise reduce oxidized nanoclusters and render them reddish [ 79 , 80 ].

Lateral flow biosensor Lateral flow biosensor is the most commonly used technology for the point of care testing [ 81 , 82 , 83 , 84 ]. A test strip consists of four parts: a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorption pad. This method uses fiber chromatography material as a solid phase to allow capillary flow of sample solution, followed by the reaction between the analyte in the sample and the target recognition molecules fixed on the nitrocellulose membrane [ 9 ] Figure 2.

Color development can be obtained through enzymatic reaction, or visually detectable materials such as gold nanoparticles. For the detection of nucleic acids, traditional lateral flow biosensor has been modified and termed nucleic acid lateral flow biosensor. In nucleic acid lateral flow biosensors, antibodies or antigens are replaced with probe DNAs that are fixed on the test zone and control zone to capture specific targets via nucleic acid hybridization.

This nucleic acid biosensor consists of i a specific capture probe, complementary to one part of the target nucleic acid and conjugated on gold nanoparticles, ii a target-hybridizing probe, immobilized on the nitrocellulose membrane test zone to capture amplified target sequence, and iii a specific nucleic acid probe on control zone that can hybridize with nanoparticle labeled probe. The hybridization on test line occurs at the presence of target, while in the absence of target sequence, the test zone does not show up.

The appearance of the control zone shows the assay works properly. This method is fast, specific, sensitive, and cost effective. With different targeting aptamers and corresponding test zones on the test strip, multiplexing assay can be developed to detect multiple pathogens [ 84 ]. The protospacer integrates into genome near the protospacer adjacent motif PAM region required as memory for future interrogation and cleavage of same invader depending on spacer-phage similarity.

On the basis of gRNAs, more literatures indicated that Cas12a, Cas13a, and Cas13b enzyme effectors require a mature crRNA for self-assembly and processing and ribonucleoprotein surveillance-dependent nuclease or DNA for interference activity [ 87 , 88 ]. These enzymes exhibit collateral cleavage of nucleic acid targets and nontarget single strands in vicinity.

Through the integration of a fluorescently labeled ssDNA reporter, a detectable signal can be obtained after cleavage. In contrast, Cas13 enzymes e. This activity can be enhanced with optimized target concentration, buffer, and crRNAs. Various diagnostic applications necessitate detection of one or more targets, and therefore with tremendous propensity of both platforms, CRISPR-Cas enzymes can detect a single or multiple targets in complex liquid biopsy samples.

Various samples suspected with Zika, dengue, and human papilloma viruses and bacteria as well as SNP and mutation discrimination have been developed. Their multiplex detection relies on reprogrammable crRNA tailing specific target sequence and enriched multiple motif fluorescent reporters.

For example, Gootenberg et al. After, the reporter is cleaved by Cas enzymes; the read-out can be achieved by high specific detection of four different quenched fluorescent reporters or using lateral flow biosensor analysis with specific antibodies against fluorescein-biotin reporters at conjugate pad and protein A as second antibody immobilized at the control line Figure 3.

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