Uses Nucleic Acid Amplification to Accurately Detect Virtually All Bacterial Species in Clinical Samples
This rapid PCR-based test uses nucleic acid amplification to quickly and accurately detect harmful bacteria from blood samples without producing false positives. Bloodstream infections (BSI) are among the most severe manifestations of bacterial disease. Bacteremia is an extreme bloodstream bacterial infection that causes symptoms such as fever, abdominal pain, and vomiting. If left untreated, it can eventually lead to sepsis, a life-threatening condition characterized by a body’s improper and overwhelming response to an infection. Each year, at least 1.7 million adults in the United States develop sepsis, resulting in the death of approximately 270,000 patients, killing more people than prostate cancer, breast cancer, and AIDs combined. There is an urgent need for accurate and reliable diagnostic tests for sepsis.
Currently, available strategies for detecting bloodstream infections require blood sample cultures. However, these procedures are often time-consuming, taking anywhere between 18 to 48 hours to yield results, show low sensitivity, and are likely to produce false positives, especially when patients have previously received antibiotics. PCR-based detection tests using highly conserved 16S rRNA sequences are a promising alternative, but the required Taq polymerase is often contaminated with bacterial DNA, again producing false positives. This problem has led companies such as BioFire Diagnostics and others, to resort to panels of much more species and genera-specific primer sets, rather than using broad-range primers that could amplify virtually all species.
Another issue is sensitivity. Testing blood directly for bacterial DNA is problematic because more than 99.9% of the DNA in the specimen is human, but sensitivity could be improved by using ribosomal RNA, of which there are generally greater than 1000 copies/bacterial cell. This would require use for reverse transcriptase (RT) to convert the 16S RNA to DNA so broad-range bacterial PCR could be performed. The problem present here is very high levels of contamination of commercially available RTs, with both bacterial 16S RNA as well as DNA.
Researchers at the University of Florida have developed a fast, highly- specific, and sensitive PCR-based test for detecting bacterial infections, overcoming the RT contamination problem. Involving the use of 16S sequences retaining universality while eliminating false positives, it readily and reliably detects all bacterial pathogens in blood samples.
Application
Rapid PCR-based test to accurately identify all pathogenic bacteria in patient blood samples
Advantages
- Amplifies nucleic acid directly from blood and other sterile tissue or fluid samples, reducing the time to complete a test
- Utilizes highly conserved 16S sequences, enabling detection of virtually all bacterial species, 90% or more, while preventing false positives
- The test can be automated, reducing hands-on time and providing high-throughput analysis in a short period of time
- It provides quantity determination of nucleic acids, enabling assessment of bacterial load
- Improves traditional blood cultures by using clinical assays, completing tests in 2 to 4 hours rather than 18 to 48 hours
Technology
This diagnostic test detects bacterial infections in blood and sterile body site samples, amplifying nucleic acids by PCR. Polymerase Chain Reaction (PCR) is a laboratory technique used to amplify small segments of DNA using the enzyme Taq polymerase. However, both the Taq polymerase and RT are generally highly contaminated with bacterial RNA and DNA, leading to false positives when using bacterial 16 S sequence-based primers. Researchers at the University of Florida have designed a set of specific 16 S sequences exhibiting a high degree of universality, effectively matching 90% of bacterial species while minimizing the occurrence of false positives. By performing PCR using these primer sequences, detection of virtually all bacterial species directly from blood samples becomes possible, increasing specificity and sensitivity and eliminating the need for traditional blood culture.
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