Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water

Bej, Asim K.; Mahbubani, Meena H.; Miller, Richard D.; DiCesare, Joseph L.; Haff, Lawrence A.; Atlas, Ronald M.

doi:10.1016/0890-8508(90)90026-v

Cited by 119 publications

(52 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In order to detect VHSV and IHNV together in one single reaction tube, a multiplex PCR was performed; however, this method appears to be less sensitive for detection and differentiation of infectious agents (especially viruses) in patient samples (Bej et al 1990). In the case of the IHNV multiplex PCR gave results similar to those of the virus isolation method 26,28).…”

Section: Discussionmentioning

confidence: 99%

Rapid and sensitive reverse transcriptase-polymerase chain reaction based detection and differential diagnosis of fish pathogenic rhabdoviruses in organ samples and cultured cells

Miller¹,

Rapp²,

U³

et al. 1998

Dis. Aquat. Org.

View full text Add to dashboard Cite

A reverse transcnptase-polymerase chain reaction (RT-PCR) assay was developed and applied to the detection and differentiation of viral haemorrhagic septicaemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) in organ samples and cultured cells, regardless of the serotype. This method was developed by selecting primer sets corresponding to highly conserved regions of the glycoprotein G-gene sequences of the 2 vlruses. The very fast RNA extraction, reverse transcnption and PCR permitted us to read the agarose gels within 7 to 9 h after samples, cultured cells and whole fish arrived, which is of great importance when there is reason to believe that VHSV or IHNV may be present This is also the first report of a large-scale field trial companng the RT-PCR assay in trout from 30 German fish farms (a total of 330 rainbow trout) with the usual virus isolation and identification method in order to evaluate the efficiency of the RT-PCR assay for general use in fish health management programs. RT-PCR followed by semi-nested PCR uslng RNA dlrectly extracted from fish tissue turned out to be the most sensitive method. It recognized 9 fish farms a s VHS-positive and 7 as IHN-positive. This is 3 VHS-and 4 IHN-farms more than detected by the traditional virus isolation method. By directly examining the tissue by means of a PCR test ~t was possible to detect viral RNA in acutely and subacutely to chronically diseased fish as well as in asyrnptomatlc VHS/IHNcarner fish Therefore, this effectlve and powerful assay for detecting VHSV and IHNV by means of PCR has great advantages compared with the presently used procedures KEY WORDS: RT-PCR . VHSV/IHNV . Differential diagnosis in organ samples INTRODUCTIONAquatic rhabdoviruses such as viral haemorrhagic septicaemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) are significant pathogens for salmonids. VHSV and IHNV often causes diseases with high mortality among salmonids, especially in rainbow trout Oncorhynchus mykiss.Since its first isolation (Jensen 1965) VHSV has been widely detected in Europe. VHSV was first isolated in 1988 in chinook salmon Oncorhynchus tshawytscha and in coho salmon 0. kisutch on the West coast of North America (Brunson et al. 1989, Hopper 1989. Virus isolations from Pacific cod Gadus macrocephalus and Alaskan Pacific herring Clupea harengus pallasi were reported in 1992 and 1993, respectively (Meyers et al. 1992(Meyers et al. , 1993. It was concluded that VHSV had been enzootic in the North Pacific region for some time (Batts et al. 1993). Even in the British Isles, thought to be VHSV-free, VHSV was isolated from a turbot Scophthalmus maximus farm in Scotland (Ross et al. 1994). Experimental infection showed that this turbot isolate was of low virulence for rainbow trout 0 Inter-Research 1998 Resale of full article not permitted

show abstract

Section: Discussionmentioning

confidence: 99%

Rapid and sensitive reverse transcriptase-polymerase chain reaction based detection and differential diagnosis of fish pathogenic rhabdoviruses in organ samples and cultured cells

Miller¹,

Rapp²,

U³

et al. 1998

Dis. Aquat. Org.

View full text Add to dashboard Cite

show abstract

“…Commercial and inhouse-developed NAATs are used in both clinical and environmental laboratories; however, the list of Legionella genes amplified is limited. The most common targets include a conserved segment of the rRNA genes for the 5S and 16S subunits, the 16S-23S spacer, and/or the macrophage inhibitor protein mip, found primarily in the genus Legionella and highly conserved in all L. pneumophila isolates (226,(332)(333)(334)(349)(350)(351)(352)(353)(354)(355)(356)(357)(358). Several studies have also examined alternative chromosomal targets, such as dotA, gyrB, dnaJ, wzm, and wzt (340).…”

Section: Nucleic Acid-based Molecular Diagnosticsmentioning

confidence: 99%

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

2015

View full text Add to dashboard Cite

SUMMARY Legionnaires' disease (LD) is an often severe and potentially fatal form of bacterial pneumonia caused by an extensive list of Legionella species. These ubiquitous freshwater and soil inhabitants cause human respiratory disease when amplified in man-made water or cooling systems and their aerosols expose a susceptible population. Treatment of sporadic cases and rapid control of LD outbreaks benefit from swift diagnosis in concert with discriminatory bacterial typing for immediate epidemiological responses. Traditional culture and serology were instrumental in describing disease incidence early in its history; currently, diagnosis of LD relies almost solely on the urinary antigen test, which captures only the dominant species and serogroup, Legionella pneumophila serogroup 1 (Lp1). This has created a diagnostic “blind spot” for LD caused by non-Lp1 strains. This review focuses on historic, current, and emerging technologies that hold promise for increasing LD diagnostic efficiency and detection rates as part of a coherent testing regimen. The importance of cooperation between epidemiologists and laboratorians for a rapid outbreak response is also illustrated in field investigations conducted by the CDC with state and local authorities. Finally, challenges facing health care professionals, building managers, and the public health community in combating LD are highlighted, and potential solutions are discussed.

show abstract

“…Therefore, we performed both a hot start and two rounds of amplification with different annealing temperatures. On the other hand, there is evidence that MPCR with targets that differ widely in size often favors amplification of the shorter target over the longer one, resulting in different amounts of amplified products (4,20). For this reason, to optimize the conditions for the MPCR analysis, we assayed different primer concentrations, template DNA preparations, and MgCl 2 concentrations.…”

mentioning

confidence: 99%

Multiplex PCR for Simultaneous Identification of Staphylococcus aureus and Detection of Methicillin and Mupirocin Resistance

et al. 2001

View full text Add to dashboard Cite

In this work, we describe a multiplex PCR assay for the detection of clinically relevant antibiotic resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. Conditions were optimized for the simultaneous detection of the 310-, 456-, and 651-bp regions of the mecA (encoding high-level methicillin resistance), ileS-2 (encoding high-level mupirocin resistance), and femB (encoding a factor essential for methicillin resistance) genes, respectively, from a single colony in a single reaction tube. The femB PCR fragment allows the specific identification of S. aureus. Validation of the method was performed using 50 human isolates of methicillin-resistant S. aureus (MRSA) and the appropriate control strains. This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.The selective pressure resulting from the extensive use of antibiotics over the last 50 years has led to the emergence of bacterial resistance and to the dissemination of resistance genes among pathogenic microorganisms (2,17,18). The progressive emergence and rapid dissemination of antibiotic resistance in staphylococci and its association with the use and consumption of antibiotics constitute a major health concern and have been considered a global crisis (7,11,16,32). Staphylococci are ubiquitous microorganisms present in the respiratory tract and on the skin of a high percentage of adults. However, several population groups are at serious risk of suffering pathogenic staphylococcal infections. Within the genus Staphylococcus, S. aureus is the causal agent of most staphylococcal infections and is associated with serious communityacquired and nosocomial diseases. Serious complications occur because of multiple-antibiotic-resistant S. aureus. The introduction of new antibiotics in the fight against staphylococcal infections has stimulated a remarkable case of bacterial evolution in the face of changing selective pressures. Thus, the use of a new drug has always been followed by the prompt appearance of new staphylococcal resistance.The first semisynthetic penicillin, namely, methicillin, was introduced in 1959 to overcome the problems that arose from the increasing prevalence of penicillinase-producing S. aureus resistant to penicillin (15). During the 1980s, methicillin-resistant S. aureus (MRSA) started to constitute a widespread human health concern (7). Methicillin resistance in S. aureus is primarily mediated by the overproduction of PBP2a, an additional altered penicillin-binding protein with low affinity for beta-lactam antibiotics. The mecA gene, the structural determinant encoding PBP2a, is therefore considered a useful molecular marker of putative methicillin resistance in S. aureus. MRSA is one of the most important pathogens that caus...

show abstract

Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water

Cited by 119 publications

References 11 publications

Rapid and sensitive reverse transcriptase-polymerase chain reaction based detection and differential diagnosis of fish pathogenic rhabdoviruses in organ samples and cultured cells

Rapid and sensitive reverse transcriptase-polymerase chain reaction based detection and differential diagnosis of fish pathogenic rhabdoviruses in organ samples and cultured cells

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

Multiplex PCR for Simultaneous Identification of Staphylococcus aureus and Detection of Methicillin and Mupirocin Resistance

Contact Info

Product

Resources

About