John M. S. Bartlett.pdf - Bio-Nica.info

Ligase Chain Reaction 143
If ligation has taken place, the biotin on the other end of the product is detected by antibiotin
conjugated to alkaline phosphatase. Alkaline phosphatase is detected by adding
methylumbelliferone phosphate that, when the phosphatase acts on the phosphate, yields a
fluorescent product that is detected by the IMx. (36). The amount of fluorescence produced
is proportional to the ligated oligonucleotides.
7. Of the many methods used to detect ligation products, automated methods such as the
IMx are the most labor efficient. Details and requirements regarding the actual use of
automated systems for LCR detection are instrument-specific and must be performed with
the manufacturers in order to ensure the validity and accuracy of the results.
3.3. Abbott LCx Commercial Automated Neisseria gonorrhoeae
Detection System
The materials listed here and the procedures described in the subsequent section are
based on what is needed for performance of the commercially sold Abbott LCx assay
for detection of N. gonorrhoeae from urogenital swabs or voided urine from men or
women. Due to the proprietary nature of the LCx technology, use of these specific
materials, including specimen transport systems and oligonucleotide primers that must
be purchased from the manufacturer in kit form is necessary for the successful performance
of this type of LCR assay. Strict adherence to the manufacturer’s instructions for
sample collection, storage, and processing is necessary for satisfactory results. The LCx
kits for C. trachomatis and M. tuberculosis follow the same general principles. Some
modifications are necessary for the M. tuberculosis assay due to the nature of the different
specimen types (respiratory secretions vs urogenital swabs or urine). It is possible to
perform the LCR assay using other commercially available ligases and methods for
detection of amplicons as described earlier, but the principles of the Abbott LCx and the
notes regarding its performance are generally relevant for any type of LCR assay.
3.3.1. Specimen Collection
1. 15–20 mL of first-void urine should be collected into a sterile plastic, preservative-free
container. It is desirable to obtain specimens from patients who have not urinated within
1 h prior to collection.
2. Swab specimens should be collected using the LCx swab collection and transport kit. For
endocervical specimens in females, excess cervical mucus should be removed prior to
sampling using the large-tipped cleaning swab provided in the collection system. When
sampling the cervix, the small-tipped swab should be inserted into the endocervix and
rotated for 15–30 s to ensure adequate sampling. In males, the small-tipped swab should
be inserted 2–4 mm into the urethral meatus and rotated for 3 to 5 s. Swabs are then
inoculated into the transport tube, broken off at the score line and then the cap is screwed
securely onto the tube.
3.3.2. Specimen Storage
1. Time and temperature conditions must be adhered to for storage and transport of specimens.
Swabs in the transport system can be stored at 2–30°C. If more than 24 h will elapse
before processing, the swabs should ideally be refrigerated at 2–8°C or frozen at –20°C
or below.
2. Urine specimens should be refrigerated immediately at 2–8°C and can be held at this
temperature for up to 4 d before processing. If longer storage is necessary, swab or urine
specimens can be frozen at –20°C or below for up to 60 d. Do not thaw urine until ready
for testing.